Collagen-localized immunomodulatory molecules and methods thereof

ABSTRACT

The present disclosure provides immunomodulatory fusion proteins comprising a collagen-binding domain operably linked to an immunomodulatory domain. The disclosure also features compositions and methods of using the same, for example, to treat cancer.

RELATED INFORMATION

This application claims the benefit of the priority date of U.S.Provisional Application No. 62/738,981, filed on Sep. 28, 2018, thecontent of which is hereby incorporated by reference in its entirety.

GOVERNMENT FUNDING

This invention was made with Government support under Grant No(s) R01CA096504 and R01 CA174795 both awarded by The National Institute ofHealth (NIH). The Government has certain rights in the invention.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format via EFS-Web and is herebyincorporated by reference in its entirety. Said ASCII copy, created Nov.22, 2019, is named “MITN-048_Sequence-Listing.txt” and is 767370Kilobytes in size.

BACKGROUND

While immunotherapy has transformed oncology with durable curativeresponses in a minority of patients, immune-related adverse events(irAEs) limit its broadest application (Michot et al. 2016, Eur JCancer, 54:139-148). It is desired to restrict the most potent immuneactivation events to tumor tissue, while sparing non-tumor healthytissue. An accepted objective of new immunotherapies is to “heat up”immunologically “cold” tumors, driving inflammation and immune cellinfiltration (Chen and Mellman 2017, Nature, 541:321-330). Varioustumor-localization approaches have been proposed: linkingimmunomodulatory agents to tumor-targeting modules in immunocytokines(Hutmacher and Neri 2018, Adv Drug Deliv Rev); masking agent activitysystemically, with tumor-localized proteolytic activation (Thomas andDaugherty 2009, Protein Sci 18:2053-2059); intratumoral injection of theagents (Singh and Overwijk 2015, Nat Commun 8:1447; Ager et al. 2017,Cancer Immunol Res 5:676-684; Bommareddy et al. 2017, Cancer J 23:40-47;Milling et al. 2017, Adv Drug Deliv Rev 114:79-101; Singh et al. 2017,Nat Commun 8:1447; Sagiv-Barfi et al. 2018, Sci Transl Med 10:eaan4488);peritumoral injection of a solid biomaterial to entrap the agent (Parket al. 2018, Sci Transl Med, 10:eaar1916); conjugation to a solidparticle (Kwong et al. 2013, Cancer Res 73:1547-1558) or conjugation ofbasic charged peptides to drive some nonspecific sticking of the agentto tumor extracellular matrix (Ishihara et al. 2017, Sci Transl Med9:eaan0401; Ishihara et al. 2018, Mol Cancer Ther 17:2399-2411). Arelated but distinct approach is to localize growth factors in tissue todrive tissue regeneration (Nishi et al. 1998, Proc Natl Acad Sci95:7018-7023; Martino et al. 2014, Science 343:885-888; Mitchell et al.2016, Acta Biomater 30:1-12).

Significant problems exist with each of the current approaches above.Immunocytokines systemically expose immune cells to the immunomodulatoryagent (Tzeng et al. 2015, Proc Natl Acad Sci 112:3320-3325). Maskingagents may be unmasked outside target tissues, and the masking agent maycomplicate manufacturing and immunogenicity. Intratumoral injectionoften leads to rapid diffusion out of the tumor compartment. Conjugationof peptides at random sites is difficult to reproduce, can negativelyimpact specific activity, doesn't fully prevent tumor exit, and createssignificant CMC issues due to the heterogeneous products of randomconjugation methods.

Accordingly, there remains a need for novel immunotherapy approaches topromote tumor-localization and increase efficacy, while preventingsystemic toxicity.

SUMMARY OF THE DISCLOSURE

The present disclosure is based, at least in part, on the discovery thatan immunomodulatory domain (e.g., cytokine, anti-immune receptorantibody, anti-tumor associated-antigen antibody, etc.) can beconjugated to a collagen-binding domain, resulting in enhancedanti-tumor efficacy relative to the unconjugated immunomodulatorydomain. Without wishing to be bound by theory, collagen localization ofan immunomodulatory domain results in enhanced anti-tumor efficacybecause T cells become entrapped in collagen-rich zones around tumors,thus making such sites desirable for targeting of immunomodulatoryagents. Almost half of human tumors exhibit an immune-excludedphenotype, wherein CD8+ T cells are apparently trapped withincollagen-rich desmoplastic stroma (Mariathasan, et al., Nature, 2018,554:544-548). Given the primary importance of CD8+ T cells inimmunotherapeutic efficacy, there is a desire to localizeimmunomodulatory agents to this collagen-rich, CD8+ T cell-richcompartment of tumors. Specificity is of significance because prioragents which utilize nonspecific electrostatic interactions in smallunstructured peptides for retention (Martino, et al., Science, 2014,343:885-888), bind promiscuously to the great majority ofnegatively-charged extracellular matrix components rather than withinthe particular collagen-rich compartment of interest. Such unstructured,positively charged peptides also lead to relatively weak retentionkinetics, wherein in some cases half of the injected conjugate payloadleaking into systemic circulation (Ishihara, et al. Mol Cancer Ther.2018, 17:2399-2411).

Accordingly, provided herein are immunomodulatory fusions to structuredproteins with specific affinity for collagen, leading to greaterretention within the particular collagen-rich compartments of interest.In some aspects described herein, the immunomodulatory fusion proteinscomprise a cytokine, wherein the collagen-binding domain increases tumorretention and prevents systemic exposure to the cytokine followingintratumoral administration in pre-clinical animal models, therebyreducing treatment-related toxicity. Furthermore, the immunomodulatoryfusion proteins have increased anti-tumor efficacy and reduced toxicitycompared to equivalent fusion proteins lacking collagen-binding domainswhen combined with one or more additional immunotherapies (e.g.,tumor-targeting antibodies, checkpoint blockade, cancer vaccines, and Tcell therapy.

As provided herein, these immunomodulatory fusion proteins demonstratedurable and systemic antitumor responses, enabling localized immunityagainst injected tumor and systemic immunity for effective treatment ofa contralateral noninjected tumor. Neoadjuvant administration of theimmunomodulatory fusion proteins also improved survival by preventingmetastases following surgical excision of residual primary tumor,further demonstrating that the immunomodulatory fusion proteins promotesystemic anti-tumor immunity. Thus, the immunomodulatory fusion proteinsof the disclosure are useful for treating metastatic tumors and/ormediating abscopal effect in therapeutic (e.g., anti-cancer) modalities.

Also provided herein, are variant collagen-binding domains that havealtered (e.g., increased or decreased) binding affinities for collagen.By disclosing a selection of variant collagen-binding domains withdifferent collagen-binding affinities, the disclosures herein provideoptions for selecting immunomodulatory fusion proteins with differentbinding affinities for collagen-rich compartments (e.g.,collagen-expressing tumors).

The collagen-binding compositions and methods provided herein allow fortumor- and payload-agnostic local targeting of active therapeutics. Thecollagen-binding compositions also demonstrate increase efficacy withconcomitant decrease in toxicity associated with systemicimmunotherapies.

In some aspects, the disclosure provides an immunomodulatory fusionprotein comprising:

(i) an immunomodulatory domain;

(ii) a collagen-binding domain, wherein the collagen-binding domainspecifically binds type I and/or type IV collagen and binds type Icollagen with a K_(D)≤500 nM, and wherein the collagen-binding domainhas an isoelectric point pI<10 and a molecular weight (MW) of ≥5 kDa;and

(iii) optionally, a linker,

wherein the immunomodulatory domain is operably linked with or withoutthe linker to the collagen-binding domain.

In some aspects, the K_(D) of the collagen-binding domain for type Iand/or type IV collagen is less than the K_(D) of the collagen-bindingdomain for an extracellular matrix component selected from fibronectin,vitronectin, osteopontin, tenascin C, or fibrinogen. In some aspects,the collagen-binding domain has a MW of about 5-100 kDa, about 10-80kDa, about 20-60 kDa, about 30-50 kDa, or about 10 kDa, about 20 kDa,about 30 kDa, about 40 kDa, about 50 kDa, about 60 kDa, about 70 kDa,about 80 kDa, about 90 kDa or about 100 kDa.

In some aspects, the immunomodulatory fusion protein comprises acollagen-binding domain comprising one or more leucine-rich repeatswhich bind collagen. In some aspects, the collagen-binding domaincomprises two, three, four, five, six, seven, eight, nine or tenleucine-rich repeats which bind collagen. In some aspects, thecollagen-binding domain comprises one or more leucine-rich repeats froma human proteoglycan Class II member of the small leucine-richproteoglycan (SLRP) family. In some aspects, the SLRP is selected fromlumican, decorin, biglycan, fibromodulin, chondroadherin, asporin,PRELP, osteoadherin/osteomodulin, opticin, osteoglycin/mimecan, podocan,perlecan, and nidogen. In some aspects, the SLRP is lumican.

In some aspects, the immunomodulatory fusion protein comprises acollagen-binding domain comprising a human SLRP. In some aspects, theSLRP is selected from lumican, decorin, biglycan, fibromodulin,chondroadherin, asporin, PRELP, osteoadherin/osteomodulin, opticin,osteoglycin/mimecan, podocan, perlecan, and nidogen. In some aspects,the SLRP is lumican. In some aspects, lumican comprises the amino acidsequence as set forth in SEQ ID NO: 107.

In some aspects, the immunomodulatory fusion protein comprises acollagen-binding domain comprising a human type I glycoprotein having anIg-like domain, or an extracellular portion thereof which bindscollagen. In some aspects, the type I glycoprotein competes with lumicanfor binding for binding to collagen type I. In some aspects, the humantype I glycoprotein is selected from LAIR1, LAIR2, and Glycoprotein IV.In some aspects, the human type I glycoprotein is LAIR1. In someaspects, the human type I glycoprotein is LAIR1 and the collagen-bindingdomain comprises amino acid residues 22-122 of the amino acid sequenceas set forth in SEQ ID NO: 98. In some embodiments, the LAIR1 is avariant comprising one or more amino acid substitutions, additions ordeletions, optionally two, three, four, five, six, seven, eight, nine,ten or more amino acid substitutions, additions or deletions relative toa LAIR1 protein comprising the amino acid sequence of SEQ ID NO: 98. Insome embodiment, the LAIR1 variant has increased binding affinity tocollagen relative to a collagen binding affinity of a LAIR1 proteincomprising the amino acid sequence of SEQ ID NO: 98. In other furtherembodiments, the LAIR1 variant has decreased binding affinity tocollagen relative to a collagen binding affinity of a LAIR1 proteincomprising the amino acid sequence of SEQ ID NO: 98.

In any of the foregoing aspects, the immunomodulatory domain comprises apolypeptide that activates, enhances or promotes a response by an immunecell. In other aspects, the immunomodulatory domain comprises apolypeptide that inhibits, reduces or suppresses a response by an immunecell.

In some aspects, the immune cell is a lymphoid cell selected from aninnate lymphoid cell, a T cell, a B cell, an NK cell, and a combinationthereof. In other aspects, the immune cell is a myeloid cell selectedfrom a monocyte, a neutrophil, a granulocyte, a mast cell, a macrophage,a dendritic cell, and a combination thereof.

In some aspects, the response by the immune cell comprises cytokineproduction, antibody production, production of antigen-specific immunecells, increased effector function and/or cytotoxicity, and acombination thereof.

In any of the foregoing aspects, the immunomodulatory domain comprisesone or more selected from a cytokine, a chemokine, an activatingligand/receptor, an inhibitory ligand/receptor, or a combinationthereof. In some aspects, the immunomodulatory domain comprises one ormore cytokines.

In some aspects, the cytokine is a human gamma common chain receptorinterleukin selected from IL-2, IL-4, IL-7, IL-9, IL-13, IL-15,IL-15/IL-15RA, IL-21, and a combination thereof. In some aspects, thecytokine is IL-2.

In some aspects, the cytokine is a human IL-12 family member selectedfrom IL-12 (p35), IL-12 (p40), IL-12(p35)/IL-12(p40), IL-23, IL-27IL-35, and a combination thereof. In some aspects, the cytokine is asingle chain fusion of IL-12(p35)/IL-12(p40).

In other aspects, the cytokine is a human IL-1 family member selectedfrom IL-1, IL-18, IL-33, and a combination thereof. In some aspects, thecytokine is IL-18.

In yet other aspects, the cytokine is selected from TNFα, INFα, IFN-γ,GM-CSF, FLT3L, G-CSF, M-CSF, and a combination thereof.

In some aspects, the immunomodulatory domain comprises one or morechemokines. In some aspects, the chemokine is selected from LIF, MIP-2,MIP-1α, MIP-1β, CXCL1, CXCL9, CXCL10, MCP-1, Eotaxin, RANTES, LIX and acombination thereof. In other aspects, the chemokine is selected fromCCL3, CCL4, CCL5, Eotaxin and a combination thereof.

In any of the foregoing aspects, the immunomodulatory domain comprisesone or more activating ligands/receptors. In some aspects, theactivating ligand/receptor is selected from a TNF superfamily, a CD28receptor superfamily, a B7 ligand family, and a T cell receptor. Inother aspects, the activating ligand/receptor is a TNF superfamilyligand selected from TNF-alpha, CD40L, 4-1BBL, OX40, and a combinationthereof. In yet other aspects, the activating ligand/receptor is a TNFsuperfamily receptor and the immunomodulatory domain comprises anantibody or antigen binding fragment thereof selected from an anti-TNFR1antibody, an anti-TNFR2 antibody, an anti-CD40 antibody, an anti-4-1BBantibody and an anti-OX40 antibody. In other aspects, the activatingligand/receptor is a CD28 superfamily member or a B7 family memberselected from ICOS ligand, CD80, and CD86, and a combination thereof. Inyet other aspects, the activating ligand/receptor is a CD28 superfamilymember and the immunomodulatory domain comprises an antibody or antigenbinding fragment thereof selected from an anti-ICOS antibody and ananti-CD28 antibody. In further aspects, the activating ligand/receptoris a T cell receptor and the immunomodulatory domain comprises anantibody or antigen binding fragment thereof selected from an anti-CD3γantibody, an anti-CD3δ antibody, an anti-CD3ζ antibody, and an anti-CD3εantibody.

In some aspects, the activating ligand/receptor is selected from a TNFsuperfamily, a CD28 receptor superfamily, a B7 ligand family, a T cellreceptor, a Killer Cell Ig-Like receptor, a Leukocyte Ig-Like receptor,a CD94/NKG2 receptor family, and an Fc receptor. In other aspects, theactivating ligand/receptor is a Killer Cell Ig-Like Receptor Ligand andthe immunomodulatory domain comprises antibody or antigen bindingfragment thereof selected from an anti-KIR 2DS1 antibody, an anti-KIR2DS2 antibody, an anti-KIR 2DS3 antibody, an anti-KIR 2DS4 antibody, ananti-KIR 2DS5 antibody and an anti-KIR 3DS1 antibody. In furtheraspects, the activating ligand/receptor is a Leukocyte Ig-Like receptorand the immunomodulatory domain comprises an anti-LIRA2 antibody orantigen binding fragment thereof. In other aspects, the activatingligand/receptor is an CD94/NKG2 receptor family member selected fromMICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5 (isoform 1) ULBP5 (isoform2), and ULBP6. In yet other aspects, the activating ligand/receptor isan CD94/NKG2 receptor family member and the immunomodulatory domaincomprises an antibody or antigen binding fragment thereof selected froman anti-CD94/NKG2D antibody, an anti-CD94/NKG2C antibody, ananti-CD94/NKG2E antibody, and an anti-CD94/NKG2H antibody. In furtheraspects, the activating ligand/receptor is an Fc receptor family memberand the immunomodulatory domain comprises an antibody or antigen bindingfragment thereof selected from anti-FcγRI antibody, an anti-FcγRIICantibody, an anti-FcγRIIIA antibody, an anti-FcγRIIIB antibody, ananti-FcεRI antibody, an anti-FcεRII antibody, an anti-FcαR antibody, andan anti-FcμR antibody.

In any of the foregoing aspects, the immunomodulatory domain comprisesone or more inhibitory ligands/receptors. In some aspects, theinhibitory ligand/receptor is selected from a CD28 receptor superfamily,a TNF superfamily, and a checkpoint inhibitor. In other aspects, theinhibitory ligand/receptor is a CD28 superfamily member and theimmunomodulatory domain comprises an antibody or antigen bindingfragment thereof selected from an anti-PD-1 antibody, an anti-PD-L1antibody, an anti-PD-L2 antibody, an anti-CTLA4 antibody. In yet furtheraspects, the inhibitory ligand/receptor is a TNF superfamily member andthe immunomodulatory domain comprises an antibody or antigen bindingfragment selected from an anti-TIGIT antibody and an anti-BTLA antibody.In some aspects, the inhibitory ligand/receptor is a checkpointinhibitor and the immunomodulatory domain comprises an antibody orantigen binding fragment selected from an anti-VISTA antibody, ananti-TIM-3 antibody, an anti-LAG-3 antibody, an anti-CD47 antibody, andan anti-SIRPα antibody.

In some aspects, the inhibitory ligand/receptor is selected from a CD28receptor superfamily, a TNF superfamily, a Siglec family, a CD94/NKG2Afamily, a Leukocyte Ig-Like receptor family, Killer Cell Ig-LikeReceptor Ligand, an Fc Receptor, an adenosine pathway molecule, and acheckpoint inhibitor. In other aspects, the inhibitory ligand/receptorcomprises a Siglec family member and the immunomodulatory domaincomprises an antibody or antigen binding fragment selected from ananti-Siglec 1 antibody, an anti-Siglec 2 antibody, an anti-Siglec 3antibody, an anti-Siglec 4a antibody, an anti-Siglec 5 antibody, ananti-Siglec 6 antibody, an anti-Siglec 7 antibody, an anti-Siglec 8antibody, an anti-Siglec 9 antibody, an anti-Siglec 10 antibody, ananti-Siglec 11 antibody and an anti-Siglec 12 antibody. In yet otheraspects, the inhibitory ligand/receptor comprises a CD94/NKG2 receptorfamily inhibitory receptor or inhibitory ligand and the immunomodulatorydomain comprises an antibody or antigen binding fragment selected froman anti-CD94/NKG2A antibody and an anti-CD94/NKG2B antibody. In someaspects, the inhibitory ligand/receptor comprises a Leukocyte Ig-LikeReceptor and the immunomodulatory domain comprises an antibody orantigen binding fragment selected from an anti-LIRB1 antibody, ananti-LIRB2 antibody, an anti-LIRB3 antibody, an anti-LIRB4 antibody. Inother aspects, the inhibitory ligand/receptor comprises a Killer CellIg-Like Receptor Ligand and the immunomodulatory domain comprisesantibody or antigen binding fragment thereof selected from an anti-KIR2DL1 antibody, an anti-KIR 2DL2 antibody, an anti-KIR 2DL3 antibody, ananti-KIR 2DL4 antibody, an anti-KIR 2DL5A antibody, an anti-KIR 2DL5Bantibody, an anti-KIR 3DL1 antibody, an anti-KIR 3DL2 antibody and ananti-KIR 3DL3 antibody. In yet other aspects, the inhibitoryligand/receptor comprises an Fc receptor and the immunomodulatory domaincomprises an anti-FcγRIIB antibody or antigen binding fragment. In someaspects, the inhibitory ligand/receptor comprises an adenosine pathwaymolecule and the immunomodulatory domain comprises an antibody orantigen binding fragment selected from an anti-CD39 antibody andanti-CD73 antibody. In other aspects, the inhibitory ligand/receptorcomprises a checkpoint inhibitor and the immunomodulatory domaincomprises an antibody or antigen binding fragment selected from is ananti-VISTA antibody, an anti-TIM-3 antibody an anti-LAG-3 antibody, ananti-CD47 antibody, and an anti-SIRPα antibody.

In any of the foregoing aspects, the immunomodulatory domain is operablylinked to the collagen-binding domain via a linker. In some aspects, thelinker is of sufficient length or mass to reduce adsorption of theimmunomodulatory domain onto collagen fibrils. In some aspects, thelinker provides sufficient molecular weight to the fusion protein reducediffusion from a tissue. In some aspects, the linker allows for stericseparation of the immunomodulatory domain from collagen fibrils topromote receptor/ligand engagement. In some aspects, the linker is ahydrophilic polypeptide comprising “N” amino acids in length, wherein1-1000, 10-900, 30-800, 40-700, 50-600, 100-500, or 200-400. In someaspects, the linker is human serum albumin or fragment thereof. In otheraspects, the linker comprises an Fc domain or a mutant Fc domain withreduced FcR interaction.

In any of the foregoing aspects, the immunomodulatory fusion protein isof sufficient mass to reduce size dependent escape by diffusion orconvection upon administration in vivo. In some aspects, the fusionprotein is ≥60 kDa. In some aspects, the immunomodulatory fusion proteinbinds type I and/or type IV collagen upon administration in vivo,thereby reducing systemic exposure of the immunomodulatory fusionprotein.

In some aspects, the disclosure provides an immunomodulatory fusionprotein comprising:

(i) at least one cytokine;

(ii) a collagen-binding domain, wherein the collagen-binding domainspecifically binds type I and/or type IV collagen and binds type Icollagen with a K_(D)≤500 nM, and wherein the collagen-binding domainhas an isoelectric point pI<10 and a molecular weight (MW) of ≥5 kDa;and

(iii) a linker, wherein the linker is a hydrophilic polypeptidecomprising “N” amino acids in length, wherein N=1-1000, 10-900, 30-800,40-700, 50-600, 100-500, or 200-400, wherein the cytokine is operablylinked via the linker to the collagen-binding domain, and wherein thefusion protein is ≥60 kDa. In some aspects, the K_(D) of thecollagen-binding domain for type I and/or type IV collagen is less thanthe K_(D) of the collagen-binding domain for an extracellular matrixcomponent selected from fibronectin, vitronectin, osteopontin, tenascinC, or fibrinogen. In some aspects, the collagen-binding domain comprisesa human SLRP selected from lumican, decorin, biglycan, fibromodulin,chondroadherin, asporin, PRELP, osteoadherin/osteomodulin, opticin,osteoglycin/mimecan, podocan, perlecan, and nidogen. In some aspects,the SLRP is lumican. In some aspects, the lumican comprises the aminoacid sequence as set forth in SEQ ID NO: 107. In other aspects, thecollagen-binding domain is selected from LAIR1, LAIR2, and GlycoproteinIV. In some aspects, the collagen-binding domain is LAIR1. In someaspects, the collagen-binding domain comprises amino acid residues22-122 of the amino acid sequence as set forth in SEQ ID NO: 98. In someembodiments, the LAIR1 is a variant comprising one or more amino acidsubstitutions, additions or deletions, optionally two, three, four,five, six, seven, eight, nine, ten or more amino acid substitutions,additions or deletions relative to a LAIR1 protein comprising the aminoacid sequence of SEQ ID NO: 98. In some embodiment, the LAIR1 varianthas increased binding affinity to collagen relative to a collagenbinding affinity of a LAIR1 protein comprising the amino acid sequenceof SEQ ID NO: 98. In other further embodiments, the LAIR1 variant hasdecreased binding affinity to collagen relative to a collagen bindingaffinity of a LAIR1 protein comprising the amino acid sequence of SEQ IDNO: 98.

In some aspects, the cytokine is a human gamma common chain receptorinterleukin selected from IL-2, IL-4, IL-7, IL-9, IL-13, IL-15,IL-15/IL-15RA, IL-21, and a combination thereof. In some aspects, thecytokine is IL-2. In some aspects, the cytokine is a human IL-12 familymember selected from IL-12 (p35), IL-12 (p40), IL-12(p35)/IL-12(p40),IL-23, IL-27, IL-35, and a combination thereof. In some aspects, thecytokine is a single chain fusion of IL-12(p35)/IL-12(p40). In someaspects, the immunomodulatory fusion protein comprises a secondcytokine. In some aspects, the second cytokine is IL-2.

In other aspects, the cytokine is a human IL-1 family member selectedfrom IL-1, IL-18, IL-33, and a combination thereof. In yet otheraspects, the cytokine is selected from TNFα, INFα, IFN-γ, GM-CSF, FLT3L,G-CSF, M-CSF, and a combination thereof.

In some aspects, the linker is of sufficient length or mass to reduceadsorption of the immunomodulatory domain onto collagen fibrils, and/orprovides sufficient molecular weight to the fusion protein reducediffusion from a tissue and/or allows for steric separation of theimmunomodulatory domain from collagen fibrils to promote receptor/ligandengagement. In some aspects, the linker is human serum albumin orfragment thereof. In other aspects, the linker comprises an Fc domain ora mutant Fc domain with reduced FcR interaction.

In some aspects, the fusion protein is of sufficient mass to reduce sizedependent escape by diffusion or convection upon administration in vivo.In some aspects, the fusion protein binds type I and/or type IV collagenupon administration in vivo, thereby reducing systemic exposure of theimmunomodulatory fusion protein.

In other aspects, the disclosure provides an immunomodulatory fusionprotein comprising:

(i) at least one chemokine;

(ii) a collagen-binding domain, wherein the collagen-binding domainspecifically binds type I and/or type IV collagen and binds type Icollagen with a K_(D)≤500 nM, and wherein the collagen-binding domainhas an isoelectric point pI<10 and a molecular weight (MW) of ≥5 kDa;and

(iii) a linker, wherein the linker is a hydrophilic polypeptidecomprising “N” amino acids in length, wherein N=1-1000, 10-900, 30-800,40-700, 50-600, 100-500, or 200-400, wherein the chemokine is operablylinked via the linker to the collagen-binding domain, and wherein thefusion protein is ≥60 kDa. In some aspects, the K_(D) of thecollagen-binding domain for type I and/or type IV collagen is less thanthe K_(D) of the collagen-binding domain for an extracellular matrixcomponent selected from fibronectin, vitronectin, osteopontin, tenascinC, or fibrinogen. In some aspects, the collagen-binding domain comprisesa human SLRP selected from lumican, decorin, biglycan, fibromodulin,chondroadherin, asporin, PRELP, osteoadherin/osteomodulin, opticin,osteoglycin/mimecan, podocan, perlecan, and nidogen. In some aspects,the SLRP is lumican. In some aspects, the lumican comprises the aminoacid sequence as set forth in SEQ ID NO: 107. In other aspects, thecollagen-binding domain is selected from LAIR1, LAIR2, and GlycoproteinIV. In some aspects, the collagen-binding domain is LAIR1. In someaspects, the collagen-binding domain comprises amino acid residues22-122 of the amino acid sequence as set forth in SEQ ID NO: 98. In someembodiments, the LAIR1 is a variant comprising one or more amino acidsubstitutions, additions or deletions, optionally two, three, four,five, six, seven, eight, nine, ten or more amino acid substitutions,additions or deletions relative to a LAIR1 protein comprising the aminoacid sequence of SEQ ID NO: 98. In some embodiment, the LAIR1 varianthas increased binding affinity to collagen relative to a collagenbinding affinity of a LAIR1 protein comprising the amino acid sequenceof SEQ ID NO: 98. In other further embodiments, the LAIR1 variant hasdecreased binding affinity to collagen relative to a collagen bindingaffinity of a LAIR1 protein comprising the amino acid sequence of SEQ IDNO: 98.

In some aspects, the chemokine is selected from LIF, MIP-2, MIP-1α,MIP-1β, CXCL1, CXCL9, CXCL10, MCP-1, Eotaxin, RANTES, LIX, and acombination thereof. In some aspects, the chemokine is selected fromCCL3, CCL4, CCL5, Eotaxin and a combination thereof.

In some aspects, the linker is of sufficient length or mass to reduceadsorption of the immunomodulatory domain onto collagen fibrils, and/orprovides sufficient molecular weight to the fusion protein reducediffusion from a tissue and/or allows for steric separation of theimmunomodulatory domain from collagen fibrils to promote receptor/ligandengagement. In some aspects, the linker is human serum albumin orfragment thereof. In other aspects, the linker comprises an Fc domain ora mutant Fc domain with reduced FcR interaction.

In some aspects, the fusion protein is of sufficient mass to reduce sizedependent escape by diffusion or convection upon administration in vivo.In some aspects, the fusion protein binds type I and/or type IV collagenupon administration in vivo, thereby reducing systemic exposure of theimmunomodulatory fusion protein.

In yet other aspects, the disclosure provides an immunomodulatory fusionprotein comprising:

(i) an agonist antibody which binds an activating ligand/receptorcomprising an Fc domain or a mutant Fc domain with reduced FcRinteraction; and

(ii) a collagen-binding domain, wherein the collagen-binding domainspecifically binds type I and/or type IV collagen and binds type Icollagen with a K_(D)≤500 nM, and wherein the collagen-binding domainhas an isoelectric point pI<10 and a molecular weight (MW) of ≥5 kDa,

wherein the collagen-binding domain is operably linked to the C-terminusof the Fc domain or mutant Fc domain. In some aspects, the K_(D) of thecollagen-binding domain for type I and/or type IV collagen is less thanthe K_(D) of the collagen-binding domain for an extracellular matrixcomponent selected from fibronectin, vitronectin, osteopontin, tenascinC, or fibrinogen. In some aspects, the collagen-binding domain comprisesa human SLRP selected from lumican, decorin, biglycan, fibromodulin,chondroadherin, asporin, PRELP, osteoadherin/osteomodulin, opticin,osteoglycin/mimecan, podocan, perlecan, and nidogen. In some aspects,the SLRP is lumican. In some aspects, the lumican comprises the aminoacid sequence as set forth in SEQ ID NO: 107. In other aspects, thecollagen-binding domain is selected from LAIR1, LAIR2, and GlycoproteinIV. In some aspects, the collagen-binding domain is LAIR1. In someaspects, the collagen-binding domain comprises amino acid residues22-122 of the amino acid sequence as set forth in SEQ ID NO: 98. In someembodiments, the LAIR1 is a variant comprising one or more amino acidsubstitutions, additions or deletions, optionally two, three, four,five, six, seven, eight, nine, ten or more amino acid substitutions,additions or deletions relative to a LAIR1 protein comprising the aminoacid sequence of SEQ ID NO: 98. In some embodiment, the LAIR1 varianthas increased binding affinity to collagen relative to a collagenbinding affinity of a LAIR1 protein comprising the amino acid sequenceof SEQ ID NO: 98. In other further embodiments, the LAIR1 variant hasdecreased binding affinity to collagen relative to a collagen bindingaffinity of a LAIR1 protein comprising the amino acid sequence of SEQ IDNO: 98.

In some aspects, the agonist antibody is selected from an anti-TNFR1antibody, an anti-TNFR2 antibody, an anti-CD40 antibody, an anti-4-1BBantibody and an anti-OX40 antibody. In other aspects, the agonistantibody is selected from an anti-ICOS antibody and an anti-CD28antibody. In some aspects, the agonist antibody is selected from ananti-CD3γ antibody, an anti-CD3δ antibody, an anti-CD3ζ antibody, and ananti-CD3ε antibody.

In some aspects, the fusion protein is of sufficient mass to reduce sizedependent escape by diffusion or convection upon administration in vivo.In some aspects, the fusion protein binds type I and/or type IV collagenupon administration in vivo, thereby reducing systemic exposure of theimmunomodulatory fusion protein.

In further aspects, the disclosure provides an immunomodulatory fusionprotein comprising:

(i) an antagonist antibody which binds an inhibitory ligand/receptorcomprising an Fc domain or a mutant Fc domain with reduced FcRinteraction; and

(ii) a collagen-binding domain, wherein the collagen-binding domainspecifically binds type I and/or type IV collagen and binds type Icollagen with a K_(D)≤500 nM, and wherein the collagen-binding domainhas an isoelectric point pI<10 and a molecular weight (MW) of ≥5 kDa,

wherein the collagen-binding domain is operably linked to the C-terminusof the Fc domain or mutant Fc domain. In some aspects, the K_(D) of thecollagen-binding domain for type I and/or type IV collagen is less thanthe K_(D) of the collagen-binding domain for an extracellular matrixcomponent selected from fibronectin, vitronectin, osteopontin, tenascinC, or fibrinogen. In some aspects, the collagen-binding domain comprisesa human SLRP selected from lumican, decorin, biglycan, fibromodulin,chondroadherin, asporin, PRELP, osteoadherin/osteomodulin, opticin,osteoglycin/mimecan, podocan, perlecan, and nidogen. In some aspects,the SLRP is lumican. In some aspects, the lumican comprises the aminoacid sequence as set forth in SEQ ID NO: 107. In other aspects, thecollagen-binding domain is selected from LAIR1, LAIR2, and GlycoproteinIV. In some aspects, the collagen-binding domain is LAIR1. In someaspects, the collagen-binding domain comprises amino acid residues22-122 of the amino acid sequence as set forth in SEQ ID NO: 98. In someembodiments, the LAIR1 is a variant comprising one or more amino acidsubstitutions, additions or deletions, optionally two, three, four,five, six, seven, eight, nine, ten or more amino acid substitutions,additions or deletions relative to a LAIR1 protein comprising the aminoacid sequence of SEQ ID NO: 98. In some embodiment, the LAIR1 varianthas increased binding affinity to collagen relative to a collagenbinding affinity of a LAIR1 protein comprising the amino acid sequenceof SEQ ID NO: 98. In other further embodiments, the LAIR1 variant hasdecreased binding affinity to collagen relative to a collagen bindingaffinity of a LAIR1 protein comprising the amino acid sequence of SEQ IDNO: 98.

In some aspects, the antagonist antibody is selected from an anti-PD-1antibody, an anti-PD-L1 antibody, an anti-PD-L2 antibody, an anti-CTLA4antibody, an anti-TIGIT antibody, an anti-BTLA antibody, an anti-VISTAantibody, an anti-TIM-3 antibody, an anti-LAG-3 antibody, an anti-CD47antibody, and an anti-SIRPα antibody.

In some aspects, the fusion protein is of sufficient mass to reduce sizedependent escape by diffusion or convection upon administration in vivo.In some aspects, the fusion protein binds type I and/or type IV collagenupon administration in vivo, thereby reducing systemic exposure of theimmunomodulatory fusion protein.

In other aspects, the disclosure provides an immunomodulatory fusionprotein comprising:

(i) human IL-2;

(ii) human lumican, human LAIR1, or human LAIR1 variant; and

(iii) a linker, wherein the linker is a hydrophilic polypeptidecomprising “N” amino acids in length, wherein N=I-1000, 10-900, 30-800,40-700, 50-600, 100-500, or 200-400, wherein IL-2 is operably linked viathe linker to lumican or LAIR1, and wherein the fusion protein is ≥60kDa.

In further aspects, the disclosure provides an immunomodulatory fusionprotein comprising:

(i) a single chain fusion of human IL-12(p35)/IL-12(p40);

(ii) human lumican, human LAIR1, or human LAIR1 variant; and

(iii) a linker, wherein the linker is a hydrophilic polypeptidecomprising “N” amino acids in length, wherein N=1-1000, 10-900, 30-800,40-700, 50-600, 100-500, or 200-400,

wherein the single chain fusion of IL-12(p35)/IL-12(p40) is operablylinked via the linker to lumican or LAIR1, and wherein the fusionprotein is ≥60 kDa.

In yet further aspects, the disclosure provides an immunomodulatoryfusion protein comprising:

(i) human CCL-3;

(ii) human lumican, human LAIR1, or human LAIR1 variant; and

(iii) a linker, wherein the linker is a hydrophilic polypeptidecomprising “N” amino acids in length, wherein N=1-1000, 10-900, 30-800,40-700, 50-600, 100-500, or 200-400,

wherein CCL-3 is operably linked via the linker to lumican or LAIR1, andwherein the fusion protein is ≥60 kDa.

In other aspects, the disclosure provides an immunomodulatory fusionprotein comprising:

(i) human CCL-4;

(ii) human lumican, human LAIR1, or human LAIR1 variant; and

(iii) a linker, wherein the linker is a hydrophilic polypeptidecomprising “N” amino acids in length, wherein N=1-1000, 10-900, 30-800,40-700, 50-600, 100-500, or 200-400,

wherein CCL-4 is operably linked via the linker to lumican or LAIR1, andwherein the fusion protein is ≥60 kDa.

In some aspects, the disclosure provides an immunomodulatory fusionprotein comprising:

(i) human CCL-5;

(ii) human lumican, human LAIR1, or human LAIR1 variant; and

(iii) a linker, wherein the linker is a hydrophilic polypeptidecomprising “N” amino acids in length, wherein N=1-1000, 10-900, 30-800,40-700, 50-600, 100-500, or 200-400,

wherein CCL-5 is operably linked via the linker to lumican or LAIR1, andwherein the fusion protein is ≥60 kDa.

In other aspects, the disclosure provides an immunomodulatory fusionprotein comprising:

(i) human Eotaxin;

(ii) human lumican, human LAIR1, or human LAIR1 variant; and

(iii) a linker, wherein the linker is a hydrophilic polypeptidecomprising “N” amino acids in length, wherein N=1-1000, 10-900, 30-800,40-700, 50-600, 100-500, or 200-400,

wherein Eotaxin is operably linked via the linker to lumican or LAIR1,and wherein the fusion protein is ≥60 kDa.

In any of the foregoing aspects, lumican comprises the amino acidsequence as set forth in SEQ ID NO: 107.

In any of the foregoing aspects, LAIR1 comprises the amino acid sequenceas set forth in SEQ ID NO: 98. In some embodiments, the LAIR1 is avariant comprising one or more amino acid substitutions, additions ordeletions, optionally two, three, four, five, six, seven, eight, nine,ten or more amino acid substitutions, additions or deletions relative toa LAIR1 protein comprising the amino acid sequence of SEQ ID NO: 98. Insome embodiment, the LAIR1 variant has increased binding affinity tocollagen relative to a collagen binding affinity of a LAIR1 proteincomprising the amino acid sequence of SEQ ID NO: 98. In other furtherembodiments, the LAIR1 variant has decreased binding affinity tocollagen relative to a collagen binding affinity of a LAIR1 proteincomprising the amino acid sequence of SEQ ID NO: 98.

In any of the foregoing aspects, the linker is of sufficient length ormass to reduce adsorption of the immunomodulatory domain onto collagenfibrils, and/or provides sufficient molecular weight to the fusionprotein reduce diffusion from a tissue and/or allows for stericseparation of the immunomodulatory domain from collagen fibrils topromote receptor/ligand engagement. In some aspects, the linker is humanserum albumin or fragment thereof. In other aspects, the linkercomprises an Fc domain or a mutant Fc domain with reduced FcRinteraction.

In some aspects, the fusion protein is of sufficient mass to reduce sizedependent escape by diffusion or convection upon administration in vivo.In some aspects, the fusion protein binds type I and/or type IV collagenupon administration in vivo, thereby reducing systemic exposure of theimmunomodulatory fusion protein.

In yet other aspects, the disclosure provides an immunomodulatory fusionprotein comprising:

(i) an agonist antibody comprising an Fc domain or a mutant Fc domainwith reduced FcR interaction, wherein the agonist antibody is selectedfrom an anti-CD3 antibody, an anti-4-1-BB antibody, an anti-CD40antibody and an anti-OX40 antibody; and

(ii) human lumican, human LAIR1, or human LAIR1 variant;

wherein lumican or LAIR1 is operably linked to the C-terminus of the Fcdomain or mutant Fc domain.

In some aspects, the fusion protein is of sufficient mass to reduce sizedependent escape by diffusion or convection upon administration in vivo.In some aspects, the fusion protein binds type I and/or type IV collagenupon administration in vivo, thereby reducing systemic exposure of theimmunomodulatory fusion protein.

In some aspects, the disclosure provides a pharmaceutical compositioncomprising an immunomodulatory fusion protein disclosed herein, and apharmaceutically acceptable carrier.

In other aspects, the disclosure provides a nucleotide sequence encodingan immunomodulatory fusion protein disclosed herein. In some aspects,the disclosure provides an expression vector comprising a nucleic aciddisclosed herein. In other aspects, the disclosure provides a celltransformed with an expression vector disclosed herein.

In another aspects, the disclosure provides a method for producing animmunomodulatory fusion protein, the method comprising maintaining acell described herein under conditions permitting expression of theimmunomodulatory fusion protein. In further aspects, the methodcomprises obtaining the immunomodulatory fusion protein.

In other aspects, the disclosure provides a method for activating,enhancing or promoting a response by an immune cell in a subject,comprising administering to a subject in need thereof, an effectiveamount of an immunomodulatory fusion protein or pharmaceuticalcomposition disclosed herein

In yet further aspects, the disclosure provides a method for inhibiting,reducing or suppressing a response by an immune cell in a subject,comprising administering to a subject in need thereof, an effectiveamount of an immunomodulatory fusion protein or pharmaceuticalcomposition disclosed herein

In any of the foregoing aspects, the immune cell is a lymphoid cellselected from an innate lymphoid cell, a T cell, a B cell, an NK cell,and a combination thereof. In other aspects, the immune cell is amyeloid cell selected from a monocyte, a neutrophil, a granulocyte, amast cell, a macrophage, a dendritic cell, and a combination thereof. Insome aspects, the response by the immune cell comprises cytokineproduction, antibody production, production of antigen-specific immunecells, increased effector function and/or cytotoxicity, and acombination thereof. In some aspects, the immune cell occurs in a tumormicroenvironment.

In other aspects, the disclosure provides a method for reducing orinhibiting tumor growth, comprising administering to a subject in needthereof, an effective amount an immunomodulatory fusion protein orpharmaceutical composition disclosed herein.

In further aspects, the disclosure provides a method for treating cancerin a subject, comprising administering to a subject in need thereof, aneffective amount of an immunomodulatory fusion protein or pharmaceuticalcomposition disclosed herein.

In any of the foregoing aspects, an anti-tumor immune response isinduced in the subject after administration of the immunomodulatoryfusion protein or the pharmaceutical composition. In some aspects, theanti-tumor immune response is a T cell response comprising theproduction of IFNγ and/or IL-2 by one or both of CD4+ T cells and CD8+ Tcells.

In any of the foregoing aspects, infiltration of immune cells into atumor microenvironment is increased after administration of theimmunomodulatory fusion protein or the pharmaceutical composition.

In any of the foregoing aspects, the quantity of T regulatory (Treg)cells is reduced in a tumor microenvironment after administration of theimmunomodulatory fusion protein or the pharmaceutical composition. Inany of the foregoing aspects, T cell exhaustion is reduced in a tumormicroenvironment after administration of the immunomodulatory fusionprotein or the pharmaceutical composition.

In any of the foregoing aspects, the immunomodulatory fusion protein orpharmaceutical composition is administered intratumorally.

In any of the foregoing aspects, the immunomodulatory fusion protein orpharmaceutical composition is administered by viral vectors,electroporation, transplantation of cells expressing theimmunomodulatory fusion protein, or replicons.

In other aspects, the disclosure provides a kit comprising a containercomprising an immunomodulatory fusion protein described herein, and anoptional pharmaceutically acceptable carrier, or a pharmaceuticalcomposition described herein, and a package insert comprisinginstructions for administration of the fusion protein or pharmaceuticalcomposition, for treating or delaying progression of cancer or reducingor inhibiting tumor growth in a subject in need thereof.

In yet further aspects, the disclosure provides a kit comprising acontainer comprising an immunomodulatory fusion protein describedherein, and an optional pharmaceutically acceptable carrier, or apharmaceutical composition described herein, and a package insertcomprising instructions for administration of the antibody orpharmaceutical composition alone or in combination with another agent,for treating or delaying progression of cancer or reducing or inhibitingtumor growth in a subject in need thereof.

In some aspects, the disclosure provides use of an immunomodulatoryfusion protein described herein, and an optional pharmaceuticallyacceptable carrier, or a pharmaceutical composition described herein,for the manufacture of a medicament for treating or delaying progressionof cancer or reducing or inhibiting tumor growth in a subject in needthereof.

In other aspects, the disclosure provides an immunomodulatory fusionprotein described herein, and an optional pharmaceutically acceptablecarrier, or a pharmaceutical composition described herein, in themanufacture of a medicament for treating or delaying progression ofcancer or reducing or inhibiting tumor growth in a subject in needthereof.

In yet further aspects, the disclosure provides an immunomodulatoryfusion protein described herein, and an optional pharmaceuticallyacceptable carrier, or a pharmaceutical composition described herein,for use as a medicament.

In other aspects, the disclosure provides a method for reducing orinhibiting tumor growth or treating cancer in a subject, the methodcomprising administering to a subject in need thereof, an effectiveamount an immunomodulatory fusion protein or pharmaceutical compositiondescribed herein, and an effective amount of a second compositioncomprising a tumor antigen-targeting antibody, or antigen-bindingfragment thereof, thereby reducing or inhibiting tumor growth ortreating cancer in the subject. In some aspects, the tumor antigen is atumor-associated antigen (TAA), a tumor-specific antigen (TSA), or atumor neoantigen. In other aspects, the tumor antigen-targeting antibodyspecifically binds human HER-2/neu, EGFR, VEGFR, CD20, CD33, or CD38.

In yet other aspects, the disclosure provides a method for reducing orinhibiting tumor growth or treating cancer in a subject, the methodcomprising administering to a subject in need thereof, an effectiveamount an immunomodulatory fusion protein or pharmaceutical compositiondescribed herein, and an effective amount of a second compositioncomprising a cancer vaccine, thereby reducing or inhibiting tumor growthor treating cancer in the subject. In some aspects, the cancer vaccineis a population of cells immunized in vitro with a tumor antigen andadministered to the subject. In other aspects, the cancer vaccine is apeptide comprising one or more tumor-associated antigens. In someaspects, cancer vaccine is an amphiphilic peptide conjugate comprising atumor-associated antigen, a lipid, and optionally a linker, wherein theamphiphilic peptide conjugate binds albumin under physiologicalconditions. In some aspects, the cancer vaccine further comprises anadjuvant.

In some aspects, the disclosure provides a method for reducing orinhibiting tumor growth or treating cancer in a subject, the methodcomprising administering to a subject in need thereof, an effectiveamount an immunomodulatory fusion protein or pharmaceutical compositiondescribed herein, and an effective amount of a second compositioncomprising an immune checkpoint inhibitor, thereby reducing orinhibiting tumor growth or treating cancer in the subject. In someaspects, the immune checkpoint inhibitor comprises an antibody orantigen binding fragment thereof which binds PD-1, PD-L1, CTLA-4, LAG3,or TIM3.

In further aspects, the disclosure provides a method for reducing orinhibiting tumor growth or treating cancer in a subject, the methodcomprising administering to a subject in need thereof, an effectiveamount an immunomodulatory fusion protein or pharmaceutical compositiondescribed herein, and an effective amount of a second compositioncomprising an adoptive cell therapy, thereby reducing or inhibitingtumor growth or treating cancer in the subject. In some aspects, theadoptive cell therapy comprises an immune effector cell comprising achimeric antigen receptor (CAR) molecule which binds to a tumor antigen.In some aspects, the CAR molecule comprises an antigen binding domain, atransmembrane domain, and an intracellular domain comprising acostimulatory domain and/or a primary signaling domain. In some aspects,the antigen binding domain binds to the tumor antigen associated withthe disease. In some aspects, the tumor antigen is selected from CD19,EGFR, Her2/neu, CD30 and BCMA. In some aspects, the immune effector cellis a T cell, such as a CD8+ T cell. In some aspects, the immune effectorcell is a natural killer (NK) cell.

In any of the foregoing methods, the immunomodulatory fusion protein orthe pharmaceutical composition are administered intratumorally. In someaspects, the immunomodulatory fusion protein or the pharmaceuticalcomposition and the second composition are administered concurrently orsequentially.

In other aspects, the disclosure provides a method for reducing orinhibiting tumor growth or treating cancer in a subject, the methodcomprising administering to a subject in need thereof, an effectiveamount of immunomodulatory fusion protein comprising:

(i) human IL-2;

(ii) human lumican, human LAIR1, or human LAIR1 variant; and

(iii) a linker, wherein the linker is a hydrophilic polypeptidecomprising “N” amino acids in length, wherein N=1-1000, 10-900, 30-800,40-700, 50-600, 100-500, or 200-400, wherein IL-2 is operably linked viathe linker to lumican or LAIR1, and wherein the fusion protein is >60kDa,

thereby reducing or inhibiting tumor growth or treating cancer in thesubject.

In some aspects, the disclosure provides a method for reducing orinhibiting tumor growth or treating cancer in a subject, the methodcomprising administering to a subject in need thereof, an effectiveamount of an immunomodulatory fusion protein comprising:

(i) a single chain fusion of human IL-12(p35)/IL-12(p40);

(ii) human lumican, human LAIR1, or human LAIR1 variant; and

(iii) a linker, wherein the linker is a hydrophilic polypeptidecomprising “N” amino acids in length, wherein N=1-1000, 10-900, 30-800,40-700, 50-600, 100-500, or 200-400,

wherein the single chain fusion of IL-12(p35)/IL-12(p40) is operablylinked via the linker to lumican or LAIR1, and wherein the fusionprotein is >60 kDa,

thereby reducing or inhibiting tumor growth or treating cancer in thesubject.

In some aspects, the disclosure provides a method for reducing orinhibiting tumor growth or treating cancer in a subject, the methodcomprising administering to a subject in need thereof, an effectiveamount of a first composition comprising an immunomodulatory fusionprotein comprising:

(i) human IL-2;

(ii) human lumican, human LAIR1, or human LAIR1 variant; and

(iii) a linker, wherein the linker is a hydrophilic polypeptidecomprising “N” amino acids in length, wherein N=1-1000, 10-900, 30-800,40-700, 50-600, 100-500, or 200-400,

wherein IL-2 is operably linked via the linker to lumican or LAIR1, andwherein the fusion protein is >60 kDa, and a second compositioncomprising an effective amount of an immunomodulatory fusion proteincomprising:

(i) a single chain fusion of human IL-12(p35)/IL-12(p40);

(ii) human lumican, human LAIR1, or; and

(iii) a linker, wherein the linker is a hydrophilic polypeptidecomprising “N” amino acids in length, wherein N=1-1000, 10-900, 30-800,40-700, 50-600, 100-500, or 200-400,

wherein the single chain fusion of IL-12(p35)/IL-12(p40) is operablylinked via the linker to lumican or LAIR1, and wherein the fusionprotein is >60 kDa,

thereby reducing or inhibiting tumor growth or treating cancer in thesubject.

In some aspects, the method further comprises administering a second (orthird, or fourth) composition comprising an effective amount of a tumorantigen-targeting antibody, or antigen-binding fragment thereof. Inother aspects, the method further comprises administering a secondcomposition comprising an effective amount of composition comprising acancer vaccine. In yet other aspects, the method further comprisesadministering a second composition comprising an effective amount of asecond composition comprising an immune checkpoint inhibitor. In someaspects, the immune checkpoint inhibitor comprises an antibody orantigen binding fragment thereof which binds PD-1, PD-L1, CTLA-4, LAG3,or TIM3.

In another aspects, the method further comprises administering a secondcomposition comprising an effective amount of a second compositioncomprising an adoptive cell therapy, thereby reducing or inhibitingtumor growth or treating cancer in the subject. In some aspects, theadoptive cell therapy comprises an immune effector cell comprising achimeric antigen receptor (CAR) molecule which binds to a tumor antigen.In some aspects, the immune effector cell is a T cell, such as a CD8+ Tcell or an NK cell.

In any of the foregoing aspects, the immunomodulatory fusion protein orthe pharmaceutical composition are administered intratumorally.

In any of the foregoing aspects, the immunomodulatory fusion protein orthe pharmaceutical composition and the second composition areadministered concurrently or sequentially.

BRIEF DESCRIPTIONS OF THE FIGURES

FIG. 1A provides a graph showing the binding of Gaussia luciferase alone(Gluc) or fused to collagen-binding polypeptides (Lumican-Gluc, ColGs3a/s3b-Gluc, ColH s3-Gluc) to collagen type I as a function ofconcentration. Binding was determined by ELISA.

FIG. 1B provides a graph showing the binding of Gaussia luciferase alone(Gluc) or fused to collagen-binding polypeptides (Lumican-Gluc, ColGs3a/s3b-Gluc, ColH s3-Gluc) to collagen type IV as a function ofconcentration. Binding was determined by ELISA.

FIG. 1C provides a graph showing the binding of His-tagged murine LAIR-1(mLAIR1-His) and His-tagged biotinylated lumican (Lwt-HIS-b) to collagentype I as a function of concentration. Binding was determined by ELISAusing an anti-HIS antibody conjugated to horseradish peroxidase (HRP).

FIG. 1D provides a graph showing the competitive binding betweenHis-tagged murine LAIR-1 (mLAIR1) and His-tagged biotinylated lumican tocollagen type I as a function of mLAIR1 concentration. Lumican bindingto collagen type I was determined by competition ELISA in the presenceof varying concentrations of mLAIR1 using Streptavidin conjugated tohorseradish perioxidate (HRP).

FIG. 2A provides a graph quantifying relative tumor fluorescence overtime of fluorescently-labeled lumican or lumican-MSA compared tofluorescently-labeled MSA following intratumoral injection inB16F10-Trp2KO tumors as determined by in vivo fluorescence imaging.

FIG. 2B provides a graph quantifying fluorescence of serum fromB16F10-Trp2KO tumor-bearing mice injected with fluorescently-labeledlumican-MSA or fluorescently-labeled MSA as a percentage of injecteddose. Serum fluorescence was determined by fluorescent imaging ofmicro-hematocrit heparin-coated tubes containing mouse blood samples.

FIG. 3A provides a Mantel-Cox survival curve of B16F10 melanomatumor-bearing mice treated with PBS (control) (i.tu.), MSA-IL2 (i.tu.),Lumican-MSA-IL2 (i.tu.), or Lumican (i.tu.). Mice (n=5 or 7 pertreatment group) were treated as indicated (arrows) on day 6 and 12.Survival statistics determined by log-rank Mantel-Cox test. Significanceindicated with **(P<0.002).

FIG. 3B provides a Mantel-Cox survival curve of B16F10 melanomatumor-bearing mice treated intratumorally with PBS (n=7, i.tu.),anti-TYRP1 antibody (TA99) (i.p.) in combination with MSA-IL2 (n=17,i.tu.), Lumican-MSA-IL2 (n=17, i.tu.), or with Lumican (n=17, i.tu.).Mice were treated as indicated (arrows) on day 6, 12 and 18. Survivalstatistics determined by log-rank Mantel-Cox test. Significanceindicated with *(P<0.03), **(P<0.002), ***(P<0.0002), ****(P<0.0001),n.s., not significant.

FIG. 3C provides a Mantel-Cox survival curve B16F10 melanomatumor-bearing mice treated with PBS (control) (i.tu.) or with acombination of anti-TYRP1 antibody (TA99) (i.p.) and Lumican-MSA-IL2administered intratumorally (i.tu.), peritumorally (peri.tu) (i.e.adjacent to the tumor), or subcutaneously near the base of the tail(s.c. tail base) Mice were treated as indicated (arrows) on day 6, 12and 18. Survival statistics determined by log-rank Mantel-Cox test.Significance indicated with *(P<0.03), **(P<0.002), ***(P<0.0002),****(P<0.0001), n.s., not significant.

FIG. 3D provides a Mantel-Cox survival curve of B16F10 melanomatumor-bearing mice treated with PBS (control) both (i.tu.) and into theinguinal tumor draining lymph node (i.tdLN), with anti-TYRP1 antibody(TA99) (i.p) in combination with Lumican-MSA-IL2 (i.tu.) and PBS(i.tdLN), or with anti-TYRP1 antibody (TA99) (i.p) in combination withLumican-MSA-IL2 (i.tdLN) and PBS (i.tu). Mice (n=7 per treatment group)were treated as indicated (arrows) on day 6 and day 12. Survivalstatistics determined by log-rank Mantel-Cox test. Significanceindicated with **(P<0.002).

FIG. 4 provides a Mantel-Cox survival curve of B16F10 melanomatumor-bearing BatF3^(−/−) or wild-type (WT) mice treated with PBS(control) (i.tu.) or anti-TYRP1 antibody (TA99) (i.p.) in combinationwith Lumican-MSA-IL2 (i.tu) and immune cell depleting or cytokineneutralizing antibodies as indicated. Mice (n=5 per treatment group)were treated as indicated on day 6, 12 and 18. Survival statisticsdetermined by log-rank Mantel-Cox test. Significance indicated with*(P<0.03), **(P<0.002), ***(P<0.0002), ****(P<0.0001), n.s., notsignificant.

FIG. 5A provides a graph quantifying IFNγ+ cells among liveCD45+CD3+CD8+ T cells derived from splenocytes, excised on day 10 ofmice (treated as described in FIG. 3B), stimulated with irradiatedB16F10 or 4T1 cells for 12 hours in the presence of brefeldin A andsubsequently stained for surface markers and intracellular IFNγ (n=5mice per treatment group). Data analyzed by one-way ANOVA with Tukey'smultiple comparison test.

FIG. 5B provides a graph quantifying mean tumor areas of thecontralateral (untreated) (left panel) and ipsilateral (treated) (middlepanel) lesions from B16F10 melanoma tumor-bearing mice treated andpercent survival (right panel) monitored over time (n=7/group). Micewere inoculated with B16F10 cells on the right flank (ipsilateral) andwith B16F10 cells on the left flank (contralateral) on day 0.Intratumoral treatments were administered to the ipsilateral tumoralongside TA99 (i.p.) on day 6 and day 12. Tumor area (mean+S.D.) of thecontralateral (untreated) and ipsilateral (treated) lesions (left) andsurvival (right) monitored over time (n=7/group). For each group, tumorarea shown until a mouse reaches the euthanasia criterion. Survivalstatistics determined by log-rank Mantel-Cox test. Significance assumedwith *, P<0.03; **, P<0.002; ***, P<0.0002; ****, P<0.0001; n.s., notsignificant.

FIG. 6A provides a graph quantifying weight change of B16F10 melanomatumor-bearing mice after treatment with PBS (i.tu.) (n=6), lumican(i.tu.) (n=7), IL12-MSA (i.tu.) (n=7), IL12-MSA-Lumican (i.tu.) (n=7),or IL12-MSA (i.p.) (n=7). Mice were treated as indicated (arrows) on day6 and day 12.

FIG. 6B provides a survival curve for mice inoculated with B16F10melanoma tumors on day 0 and treated with PBS (control), lumican(i.tu.), IL12-MSA (i.tu), IL12-MSA (i.p.) or IL12-MSA-Lumican (i.tu) ondays 6 and 12.

FIG. 7 provides graphs depicting weight change from baseline (leftpanel) and corresponding survival over time (right panel) of B16F10tumor-bearing mice treated with intratumoral (i.tu.) injections of PBS(n=5), MSA-IL2 and IL12-MSA (n=5), or Lumican-MSA-IL2 andIL12-MSA-Lumican (n=5) on day 5 and day 11. Arrows indicate time oftreatment. Survival statistics determined by log-rank Mantel-Cox test.Significance indicated with *(P<0.03), **(P<0.002), ***(P<0.0002),****(P<0.0001), n.s., not significant. FIG. 8A provides a Mantel-Coxsurvival curve of B16F10 melanoma tumor-bearing BatF3^(−/−) or wild-type(WT) mice treated with PBS (control) (i.tu.) or Lumican-MSA-IL2 (i.tu.)in combination with IL12-MSA-Lumican (i.tu.) and immune cell depletingor cytokine neutralizing antibodies as indicated. Mice (n=5 pertreatment group) were treated as indicated on day 6, 12 and 18. Survivalstatistics determined by log-rank Mantel-Cox test. Significanceindicated with *(P<0.03), **(P<0.002), ***(P<0.0002), ****(P<0.0001),n.s., not significant.

FIG. 8B provides a Mantel-Cox survival curve of B16F10 melanomatumor-bearing BatF3−/− or wild-type (WT) mice treated with PBS (control)(i.tu.) or Lumican-MSA-IL2 (i.tu.) in combination with IL12-MSA-Lumican(i.tu.) and immune cell depleting antibodies as indicated. Mice (n=5 pertreatment group) were treated as indicated on day 6, 12 and 18. Survivalstatistics determined by log-rank Mantel-Cox test. Significanceindicated with *(P<0.03), **(P<0.002), ***(P<0.0002), ****(P<0.0001),n.s., not significant.

FIG. 8C provides a heat map showing the fold change of immune cells intumor infiltrates from B16F10 melanoma tumor-bearing mice treatedintratumorally with a combination of IL12-MSA-Lumican andLumican-MSA-IL2 (Lumican versions) or a combination of IL12-MSA+MSA-IL2(MSA versions) of relative to treatment with PBS.

FIGS. 8D-8E provide graphs quantifying tumor-infiltrating CD8+ T cellsisolated from B16F10 melanoma tumor-bearing mice on day 11 post-tumorcell injection (FIG. 8D) and their corresponding median fluorescenceintensity (MFI) of surface PD-1 (FIG. 8E), after treatment as in FIG. 8Con day 5 post-tumor cell injection.

FIG. 9 provides graphs depicting weight change from baseline (leftpanel) and corresponding survival over time (right panel) of B16F10melanoma tumor-bearing mice treated with intratumoral (i.tu.) injectionsof PBS (n=5), anti-PD-1 antibody in combination with MSA-IL2 andIL12-MSA (n=5), or anti-PD-1 antibody in combination withLumican-MSA-IL2 and IL12-MSA-Lumican (n=5) on day 5 and day 11. Arrowsindicate time of treatment. Weight change and comparison statisticsdetermined by one-way ANOVA with Tukey's multiple comparison test.Survival statistics determined by log-rank Mantel-Cox test. Significanceindicated with *(P<0.03), **(P<0.002), ***(P<0.0002), ****(P<0.0001),n.s., not significant.

FIGS. 10A-10B provides a graph depicting tumor area (left panel) andpercent survival (right panel) of EMT6 tumor-bearing mice (FIG. 10A) orMC38 tumor-bearing mice (FIG. 10B) and treated as indicated (arrows) onday 5, 11, and 17 as indicated (arrows). Weight change and comparisonstatistics determined by one-way ANOVA with Tukey's multiple comparisontest. Survival statistics determined by log-rank Mantel-Cox test.Significance indicated with *, P<0.03; **, P<0.002; ***, P<0.0002; ****,P<0.0001; n.s., not significant.

FIG. 11 provides graphs depicting weight change from baseline (left,mean+S.D.), corresponding tumor area (middle, mean+S.D.) and survival(right) of B16F10 melanoma tumor-bearing mice treated with intratumoral(i.tu.) injections of PBS (n=12) or IL-12 (n=10 for IL12-MSA; n=10 forIL12-MSA-Lumican), or cancer vaccine (n=7) alone, or cancer vaccine andIL12 (n=7 for IL12-MSA; n=7 for IL12-MSA-Lumican) on days 5, 11 and 17as indicated (arrows). Tumor area shown until a mouse reaches theeuthanasia criterion (>100 mm²). Weight change statistics, shown withinplot, determined by one-way ANOVA with Tukey's multiple comparison test.Survival statistics, adjacent to the legend, determined by log-rankMantel-Cox test. Significance assumed with *, P<0.03; **, P<0.002; ***,P<0.0002; ****, P<0.0001; n.s., not significant

FIG. 12 provides graphs depicting weight change from baseline (left,mean+S.D.), corresponding tumor area (middle, mean+S.D.) and percentsurvival (right) of B16F10 melanoma tumor-bearing mice treated withintratumoral (i.tu.) injections of PBS (n=11) or IL-12 (n=9 forIL12-MSA; n=5 for IL12-MSA-Lumican), or CAR-T (n=11) alone, or CAR-T andIL12 (n=9 for IL12-MSA; n=5 for IL12-MSA-Lumican) on days 5 and 11 asindicated (arrows). Mice were inoculated with B16F10 cells on day 0 andlymphodepleted by total body irradiation on day 4. CAR-T treatments wereadministered in a single bolus tail vein injection (i.v.) on day 5.Tumor area shown until a mouse reaches the euthanasia criterion (>100mm²). Weight change statistics, shown within plot, determined by one-wayANOVA with Tukey's multiple comparison test. Survival statistics,adjacent to the legend, determined by log-rank Mantel-Cox test.Significance assumed with *, P<0.03; **, P<0.002; ***, P<0.0002; ****,P<0.0001; n.s., not significant

FIG. 13 provides graphs depicting total body weight change duringneoadjuvant treatment (left), primary tumor growth and weight (middle)and survival (right) of 4T1 mammary carcinoma tumor-bearing mice treatedwith intratumoral (i.tu.) injections of IL-12 (n=5 for IL12; n=5 forIL12-MSA-Lumican) and intraperitoneal (i.p.) injection of anti-PD-1 onday 7 and 13. Arrows indicate time of treatment and cross indicates timeof surgery. Mice were inoculated 4T1-Luc cells in the mammary fat pad onday 0. Neoadjuvant therapy was administered on day 7 and 13 and theprimary tumors was surgically excised on day 16. Post-operation micewere monitored by in vivo imaging (IVIS) for metastases. For each group,tumor area shown until the primary tumor is excised. Weight changestatistics, shown within plot, determined by one-way ANOVA with Tukey'smultiple comparison test. Survival statistics, adjacent to the legend,determined by log-rank Mantel-Cox test. Significance assumed with *,P<0.03; **, P<0.002; ***, P<0.0002; ****, P<0.0001; n.s., notsignificant

FIG. 14A provides a graph depicting mean tumor area of 4T1 mammarycarcinoma tumor-bearing mice treated intratumorally with Lumican-GLuc ora combination of Lumican-CCL3, Lumican-CCL4 and Lumican-CCL on day 7 andday 13. Administration of IFNα intraperitoneally occurred on day 9 andday 15. Tumor growth (mean+SEM) monitored over time every other day.

FIG. 14B provides a graph depicting mean tumor area of B16F10 melanomatumor-bearing mice treated intratumorally with Lumican-GLuc or acombination of Lumican-CCL3, Lumican-CCL4 and Lumican-CCL5 on day 7 andday 13. Administration of IFNα intraperitoneally on day 9 and day 15.Tumor growth (mean+SEM) monitored over time every other day.

FIG. 14C provides a graph depicting the effect of various concentrationsof fusion proteins Lumican-GLuc (Lum GLuc), Lumican-CCL3 (Lum CCL3),Lumican-CCL5 (Lum CCL5) on the proliferation of 4T1 breast tumor cellsin vitro. Proliferation was determined by measurement of WST-1proliferation reagent by absorbance at 450 nm.

FIG. 14D provides a graph depicting the effect of various concentrationsof fusion proteins Lumican-GLuc (Lum GLuc), Lumican-CCL3 (Lum CCL3),Lumican-CCL5 (Lum CCL5) on the proliferation of B16F10 melanoma tumorcells in vitro. Proliferation was determined by measurement of WST-1proliferation reagent by absorbance at 450 nm.

FIG. 15 provides a graph depicting mean tumor areas of tumor lesions inB16F10 melanoma tumor-bearing mice treated with a cancer vaccineadministered subcutaneously (s.c.) at the tail base with a prime on day5 and boosts on day 11 and 17 post-tumor cell injection. Cancer vaccinewas administered alone or in combination with CCL11-lumican, TNFα, IFNγ,or Lumican, as indicated, administered intratumorally on days 11, 17,23, and 29. Tumor area (mean+SD) was measured over time every other day.

FIGS. 16A-16B provides a graph depicting individual tumor areas of tumorlesions in B16F10 melanoma tumor-bearing mice treated with atumor-targeting antibody 2.5F-Fc (i.p.) and MSA-IL2 (i.p.) on days 5,11, and 17 post-tumor cell injection in combination with either lumican(Lwt) (i.tu.) (FIG. 16B) or CCL11-lumican (11L) (i.tu.) (FIG. 16A)administered on days 5 and 11. Tumor area monitored over time everyother day.

FIG. 17A provides a graph showing the binding of agonistantibody-lumican fusion proteins to collagen type I as a function ofconcentration. Binding was determined by ELISA.

FIG. 17B provides a graph quantifying the in vivo fluorescence of amouse anti-FITC antibody (4420) alone or fused to Lumicanfluorescently-labeled with Alexa Fluor® 647 after intratumoral injectioninto 4T1 tumors of mic over time as determined by in vivo fluorescenceimaging. Measurement of in vivo fluorescence is provided in units oftotal radiant efficiency (p/s)/(μW/cm2).

FIG. 18A provides a graph showing the binding of a subset of His-taggedlumican-IgG binding fusion proteins, as indicated, to collagen type I(left panel) or collagen IV (right panel) as a function ofconcentration. Binding was determined by ELISA.

FIG. 18B provides a graph depicting the binding of His-taggedlumican-IgG binding fusion proteins to mouse IgG2a isotype control(Clone C1.18.4) as a function of concentration. Binding was determinedby ELISA. Anti-His (Clone ab1187) was used to detect each construct.

FIG. 19 provides 3D microscopy images of mouse omental tissue fromOVCA433 human ovarian tumor-bearing mice showing specific accumulationof Alexa Fluor 647-labeled lumican (yellow) around RFP-expressingOVCA433 human ovarian tumor cell microcolonies (Red) in the mouseomental tissue, with collagen imaged by SHG microscopy in grey. Labeledlumican was injected intraperitoneally in tumor-bearing mice.

FIG. 20A provides graphs depicting the expression of IL-12 fusionproteins alone or fused to a fluorescent protein (mCherry), asindicated, from a self-replicating RNA in B16F10 cells as determined byflow cytometry.

FIG. 20B provides a graph depicting the expression of IL-12 fusionproteins alone or fused to a fluorescent protein (mCherry), as indicted,from a self-replicating RNA in B16F10 cells as determined by an IL-12ELISA.

FIG. 21A provides a graph showing tumor volume (mean+SD) oftumor-bearing mice treated either with an intratumoral injection of PBS(n=4) or with intratumoral collagen-anchoring cytokines Lumican-MSA-IL2and IL12-MSA-Lumican and intraperitoneal TA99 and anti-PD-1 (n=5) ondays 25, 31, 37, 43, 49, 55, and 61. For each group, tumor volume shownuntil a mouse reaches the euthanasia criterion (>1200 mm³).

FIG. 21B provides a Mantel-Cox survival curve of tumor-bearing micetreated with intratumoral PBS (n=10), with intratumoral Lumican-MSA-IL2and IL12-MSA-Lumican and intraperitoneal TA99 and anti-PD-1 (n=14), withintratumoral Lumican-MSA-IL2 and IL12-MSA-Lumican and intraperitonealanti-PD-1 (n=10), with intratumoral MSA-IL2 and IL12-MSA andintraperitoneal TA99 and anti-PD-1 (n=9), or with intratumoral MSA-IL2and IL12-MSA and intraperitoneal anti-PD-1 (n=8) on days 25, 31, 37, 43,49, 55, and 61. Arrowheads indicate times of treatment. Overall survivalgraph enumerates mice that succumbed to tumor burden (>1200 mm³) or totreatment-related weight loss (>20%); the latter is indicated by a blue“x” for each mouse. Survival was compared by log-rank Mantel-Cox test.*P<0.03, ***P<0.0002, ****P<0.0001.

FIG. 22A provides a schematic for measuring LAIR binding capacity inB16F10 tumors.

FIG. 22B provides a graph showing the weight of an excised tumor and itsextracellular matrix.

FIG. 22C provides a graph showing the hydroxyproline content of theB16F10 cell fraction compared to matrix fraction.

FIG. 22D provides a graph showing depletion of LAIR-fluorescence as aB16F10 derived-matrix fraction was placed in a 1 mL of AF647-labeledLAIR.

FIG. 22E provides a graph showing the correlation between the matrixfraction hydroxyproline content and the LAIR-binding capacity ofB16F10-derived matrix fraction.

FIG. 23A provides a graph showing tumor area of B16F10 melanoma tumors(1×10⁶ cells inoculated on day 0) treated with PBS control (i.tu) (n=5)or LAIR-MSA-IL2 (i.tu) TA99 (i.p.) (n=7) on days 6, and 13.

FIG. 23B provides a Mantel-Cox survival curve of B16F10 melanomatumor-bearing mice (1×10⁶ cells inoculated on day 0) treated with PBScontrol (i.tu) (n=5) or LAIR-MSA-IL2 (i.tu)+TA99 (i.p.) (n=7) on days 6and 13.

FIG. 24A provides a sequence alignment of low affinity collagen binders,LAIR. 30.w.A, LAIR.30.w.B, LAIR.30.w.C, and LAIR.30.w. D, compared towild-type LAIR (LAIR).

FIGS. 24B-E provide depictions of the crystal structures of wild-typeLAIR (PDB 4ETY) shown as a gray ribbon, with selected mutated amino acidresidues of low affinity collagen binders, LAIR.30.w.A (FIG. 24B),LAIR.30.w.B (FIG. 24C), LAIR.30.w.C (FIG. 24D), and LAIR.30.d. D (FIG.24E), highlighted as bonded spheres.

FIG. 24F provides a graph showing binding of WT LAIR, WT LAIR-MSA,MSA-IL-2 (non-specific binding control), and mutant LAIR-MSA fusions tocollagen type 1 in an ELISA assay (n=2). The binding-affinity (Kd) ofeach LAIR construct, calculated on a non-linear one-site binding fit, isalso shown.

FIG. 25A provides a sequence alignment of low affinity collagen binders,LAIR.30.w.E and LAIR.30.w.F compared to wild-type LAIR.

FIGS. 25B-C provide depictions of the crystal structures of wild-typeLAIR (PDB 4ETY) shown as a gray ribbon, with selected mutated amino acidresidues of low affinity collagen binders, LAIR.30.w.E (FIG. 25B) andLAIR.30.w. F (FIG. 25C), highlighted as bonded spheres.

FIG. 25D provides a graph showing binding of WT LAIR, WT LAIR-MSA,MSA-IL-2 (non-specific binding control), and mutant LAIR-MSA fusions tocollagen type 1 in an ELISA assay (n=2). The binding-affinity (Kd) ofeach LAIR construct, modeled on a non-linear one-site binding fit, isalso shown.

FIG. 26A provides a sequence alignment of high affinity collagen binder,LAIR.30.2.K1.B, compared to wild-type LAIR.

FIG. 26B provides a depiction of the crystal structure of wild-type LAIR(PDB 4ETY) shown as a gray ribbon, with selected mutated amino acidresidues of high affinity collagen binder, LAIR.30.2.K1.B, highlightedas bonded spheres.

FIGS. 26C-D provide flow cytometry plots showing CRP-XL-biotin binding(Strepravidin-AF647) versus protein display (goat anti-chicken AF488) ofyeast bearing wild-type LAIR (black) or LAIR30.2.K1.B (cyan) incubatedin either 100 nM (FIG. 26C) or 0.01 nM (FIG. 26D) of CRP-XL-biotin.

FIGS. 26E-F provide flow cytometry plots showing remaining surfaceCRP-XL-biotin signal (Streptatividn-AF647) versus protein display (goatanti-chicken AF488) of yeast bearing LAIR30.2.K1.B (FIG. 26E) orwild-type LAIR (FIG. 26F) at different time points (0 hours, 16 hoursand 40 hours of competition) after competition with an excess ofnon-biotinylated CRP-XL.

FIG. 26G provides a graph showing the median fluorescence intensity ofbound CRP-XL-biotin over time in the kinetic dissociation experimentdepicted in FIGS. 26E-F. The estimated off-rates, modeled on a one-phaseexponential decay fit, are also shown.

DETAILED DESCRIPTION

Provided herein are immunomodulatory fusion proteins comprising animmunomodulatory domain operably linked to a collagen-binding domain.Such fusion proteins localize the immunomodulatory domain (e.g.,cytokine, antibody), such that it is not systemically disseminated.Systemic dissemination of an immunomodulatory domain can result inreduced efficacy due to rapid clearance from the site of interest (e.g.,tumor), and/or toxicity due to effects on non-target cells outside thetumor. Accordingly, the linking of an immunomodulatory domain to acollagen-binding protein localizes the immunomodulatory domain toprevent systemic dissemination, thereby maintaining the immunomodulatorydomain at the site of interest and reducing potential off-target effectsthat could lead to toxicity.

Definitions

Terms used in the claims and specification are defined as set forthbelow unless otherwise specified. In the case of direct conflict with aterm used in a parent provisional patent application, the term used inthe instant application shall control.

It must be noted that, as used in the specification and the appendedclaims, the singular forms “a,” “an” and “the” include plural referentsunless the context clearly dictates otherwise.

As used herein, “about” will be understood by persons of ordinary skilland will vary to some extent depending on the context in which it isused. If there are uses of the term which are not clear to persons ofordinary skill given the context in which it is used, “about” will meanup to plus or minus 10% of the particular value.

As used herein, the term “agonist” refers to any molecule (e.g., anantibody or antigen binding fragment thereof) that partially or fullypromotes, increases, or activates a biological activity of a nativepolypeptide disclosed herein. Suitable agonist molecules specificallyinclude agonist antibodies or antibody fragments, fragments or aminoacid sequence variants of native polypeptides, peptides, antisenseoligonucleotides, small organic molecules, etc. In some embodiments,activation in the presence of the agonist is observed in adose-dependent manner. In some embodiments, the measured signal (e.g.,biological activity) is at least about 5%, at least about 10%, at leastabout 15%, at least about 20%, at least about 25%, at least about 30%,at least about 35%, at least about 40%, at least about 45%, at leastabout 50%, at least about 55%, at least about 60%, at least about 65%,at least about 70%, at least about 75%, at least about 80%, at leastabout 85%, at least about 90%, at least about 95%, or at least about100% higher than the signal measured with a negative control undercomparable conditions. Also disclosed herein, are methods of identifyingagonists suitable for use in the methods of the disclosure. For example,these methods include, but are not limited to, binding assays such asenzyme-linked immuno-absorbent assay (ELISA), Forte Bio© systems, andradioimmunoassay (RIA). These assays determine the ability of an agonistto bind the polypeptide of interest (e.g., a receptor or ligand) andtherefore indicate the ability of the agonist to promote, increase oractivate the activity of the polypeptide. Efficacy of an agonist canalso be determined using functional assays, such as the ability of anagonist to activate or promote the function of the polypeptide. Forexample, a functional assay may comprise contacting a polypeptide with acandidate agonist molecule and measuring a detectable change in one ormore biological activities normally associated with the polypeptide. Thepotency of an agonist is usually defined by its EC₅₀ value(concentration required to activate 50% of the agonist response). Thelower the EC₅₀ value the greater the potency of the agonist and thelower the concentration that is required to activate the maximumbiological response.

The term “albumin” refers to a protein having the same, or very similarthree dimensional structure as human albumin (SEQ ID NO: 42) and havinga long serum half-life. Exemplary albumin proteins include human serumalbumin (HSA; SEQ ID NOs: 42 and 43), primate serum albumin (such aschimpanzee serum albumin), gorilla serum albumin or macaque serumalbumin, rodent serum albumin (such as hamster serum albumin), guineapig serum albumin, mouse serum albumin and rat serum albumin, bovineserum albumin (such as cow serum albumin), equine serum albumin (such ashorse serum albumin or donkey serum albumin), rabbit serum albumin, goatserum albumin, sheep serum albumin, dog serum albumin, chicken serumalbumin and pig serum albumin.

The term “ameliorating” refers to any therapeutically beneficial resultin the treatment of a disease state, e.g., cancer, includingprophylaxis, lessening in the severity or progression, remission, orcure thereof.

“Amino acid” refers to naturally occurring and synthetic amino acids, aswell as amino acid analogs and amino acid mimetics that function in amanner similar to the naturally occurring amino acids. Naturallyoccurring amino acids are those encoded by the genetic code, as well asthose amino acids that are later modified, e.g., hydroxyproline,γ-carboxyglutamate, and 0-phosphoserine. Amino acid analogs refers tocompounds that have the same basic chemical structure as a naturallyoccurring amino acid, i.e., an a carbon that is bound to a hydrogen, acarboxyl group, an amino group, and an R group, e.g., homoserine,norleucine, methionine sulfoxide, methionine methyl sulfonium. Suchanalogs have modified R groups {e.g., norleucine) or modified peptidebackbones, but retain the same basic chemical structure as a naturallyoccurring amino acid. Amino acid mimetics refers to chemical compoundsthat have a structure that is different from the general chemicalstructure of an amino acid, but that function in a manner similar to anaturally occurring amino acid.

Amino acids can be referred to herein by either their commonly knownthree letter symbols or by the one-letter symbols recommended by theIUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise,can be referred to by their commonly accepted single-letter codes.

An “amino acid substitution” refers to the replacement of at least oneexisting amino acid residue in a predetermined amino acid sequence (anamino acid sequence of a starting polypeptide) with a second, different“replacement” amino acid residue. An “amino acid insertion” refers tothe incorporation of at least one additional amino acid into apredetermined amino acid sequence. While the insertion will usuallyconsist of the insertion of one or two amino acid residues, larger“peptide insertions,” can also be made, e.g. insertion of about three toabout five or even up to about ten, fifteen, or twenty amino acidresidues. The inserted residue(s) may be naturally occurring ornon-naturally occurring as disclosed above. An “amino acid deletion”refers to the removal of at least one amino acid residue from apredetermined amino acid sequence.

As used herein, the term “antagonist” refers to any molecule (e.g.,antibody or antigen-binding fragment thereof) that partially or fullyblocks, inhibits, or neutralizes a biological activity of a nativepolypeptide disclosed herein. Suitable antagonist molecules specificallyinclude antagonist antibodies or antibody fragments, fragments or aminoacid sequence variants of native polypeptides, peptides, antisenseoligonucleotides, small organic molecules, etc. In some embodiments,inhibition in the presence of the antagonist is observed in adose-dependent manner. In some embodiments, the measured signal (e.g.,biological activity) is at least about 5%, at least about 10%, at leastabout 15%, at least about 20%, at least about 25%, at least about 30%,at least about 35%, at least about 40%, at least about 45%, at leastabout 50%, at least about 55%, at least about 60%, at least about 65%,at least about 70%, at least about 75%, at least about 80%, at leastabout 85%, at least about 90%, at least about 95%, or at least about100% lower than the signal measured with a negative control undercomparable conditions. Also disclosed herein, are methods of identifyingantagonists suitable for use in the methods of the disclosure. Forexample, these methods include, but are not limited to, binding assayssuch as enzyme-linked immuno-absorbent assay (ELISA), Forte Bio©systems, and radioimmunoassay (RIA). These assays determine the abilityof an antagonist to bind the polypeptide of interest (e.g., a receptoror ligand) and therefore indicate the ability of the antagonist toinhibit, neutralize or block the activity of the polypeptide. Efficacyof an antagonist can also be determined using functional assays, such asthe ability of an antagonist to inhibit the function of the polypeptideor an agonist. For example, a functional assay may comprise contacting apolypeptide with a candidate antagonist molecule and measuring adetectable change in one or more biological activities normallyassociated with the polypeptide. The potency of an antagonist is usuallydefined by its IC₅₀ value (concentration required to inhibit 50% of theagonist response). The lower the IC₅₀ value the greater the potency ofthe antagonist and the lower the concentration that is required toinhibit the maximum biological response.

As used herein, the term “antibody” refers to a whole antibodycomprising two light chain polypeptides and two heavy chainpolypeptides. Whole antibodies include different antibody isotypesincluding IgM, IgG, IgA, IgD, and IgE antibodies. The term “antibody”includes a polyclonal antibody, a monoclonal antibody, a chimerized orchimeric antibody, a humanized antibody, a primatized antibody, adeimmunized antibody, and a fully human antibody. The antibody can bemade in or derived from any of a variety of species, e.g., mammals suchas humans, non-human primates (e.g., orangutan, baboons, orchimpanzees), horses, cattle, pigs, sheep, goats, dogs, cats, rabbits,guinea pigs, gerbils, hamsters, rats, and mice. The antibody can be apurified or a recombinant antibody.

As used herein, the term “antibody fragment,” “antigen-bindingfragment,” or similar terms refer to a fragment of an antibody thatretains the ability to bind to a target antigen(s) and promote, induce,and/or increase the activity of the target antigen. Such fragmentsinclude, e.g., a single chain antibody, a single chain Fv fragment(scFv), an Fd fragment, an Fab fragment, an Fab′ fragment, or an F(ab′)2fragment. An scFv fragment is a single polypeptide chain that includesboth the heavy and light chain variable regions of the antibody fromwhich the scFv is derived. In addition, intrabodies, minibodies,triabodies, and diabodies are also included in the definition ofantibody and are compatible for use in the methods described herein.See, e.g., Todorovska et al. (2001) J Immunol Methods 248(1):47-66;Hudson and Kortt (1999) J Immunol Methods 231(1):177-189; Poljak (1994)Structure 2(12):1121-1123; Rondon and Marasco (1997) Annual Review ofMicrobiology 51:257-283, the disclosures of each of which areincorporated herein by reference in their entirety.

As used herein, the term “antibody fragment” also includes, e.g., singledomain antibodies such as camelized single domain antibodies. See, e.g.,Muyldermans et al. (2001) Trends Biochem Sci 26:230-235; Nuttall et al.(2000) Curr Pharm Biotech 1:253-263; Reichmann et al. (1999) J ImmunolMeth 231:25-38; PCT application publication nos. WO 94/04678 and WO94/25591; and U.S. Pat. No. 6,005,079, all of which are incorporatedherein by reference in their entireties. In some embodiments, thedisclosure provides single domain antibodies comprising two VH domainswith modifications such that single domain antibodies are formed.

The “B7 family” refers to activating and inhibitory ligands. The B7family encompasses at least activating ligands B7-1 and B7-2, andinhibitory ligands B7-H1, B7-H2, B7-H3 and B7-H4. B7-1 and B7-2 bind toCD28, B7-H1 (i.e., PD-L1) binds to PD-1, and B7-H2 binds to ICOS. B7-H3and B7-H4 bind unknown receptors. Further, B7-H3 and B7-H4 have beenshown to be upregulated on tumor cells and tumor infiltrating cells. Thecomplete hB7-H3 and hB7-H4 sequence can be found under GenBank AccessionNos. Q5ZPR3 and AAZ17406 (SEQ ID NOs: 49 and 50) respectively.

As used herein, the term “chimeric antigen receptor (CAR)” refers to anartificial transmembrane protein receptor comprising (i) anextracellular domain capable of binding to at least one predeterminedCAR ligand or antigen, or a predetermined CAR ligand and an antigen,(ii) an intracellular segment comprising one or more cytoplasmic domainsderived from signal transducing proteins different from the polypeptidefrom which the extracellular domain is derived, and (iii) atransmembrane domain. The “chimeric antigen receptor (CAR)” is sometimescalled a “chimeric receptor”, a “T-body”, or a “chimeric immune receptor(CIR).”

The phrase “CAR ligand” used interchangeably with “CAR antigen” meansany natural or synthetic molecule (e.g., small molecule, protein,peptide, lipid, carbohydrate, nucleic acid) or part or fragment thereofthat can specifically bind to a CAR (e.g., the extracellular domain of aCAR). In some embodiments, the CAR ligand is a tumor-associated antigen,or fragment thereof. In some embodiments, the CAR ligand is a tag.

The “intracellular signaling domain” means any oligopeptide orpolypeptide domain known to function to transmit a signal causingactivation or inhibition of a biological process in a cell, for example,activation of an immune cell such as a T cell or a NK cell. Examplesinclude ILR chain, CD28 and/or CD3.

As used herein, “cancer antigen” refers to (i) tumor-specific antigens,(ii) tumor-associated antigens, (iii) cells that express tumor-specificantigens, (iv) cells that express tumor-associated antigens, (v)embryonic antigens on tumors, (vi) autologous tumor cells, (vii)tumor-specific membrane antigens, (viii) tumor-associated membraneantigens, (ix) growth factor receptors, (x) growth factor ligands, and(xi) any other type of antigen or antigen-presenting cell or materialthat is associated with a cancer.

As used herein, “cancer vaccine” refers to a treatment that induces theimmune system to attack cells with one or more tumor associatedantigens. The vaccine can treat existing cancer (e.g., therapeuticcancer vaccine) or prevent the development of cancer in certainindividuals (e.g., prophylactic cancer vaccine). The vaccine createsmemory cells that will recognize tumor cells with the antigen andtherefore prevent tumor growth.

As used herein, the term “chemokine” refers to a member of the family ofsmall cytokines, or signaling proteins, that induce directed chemotaxis.Chemokines are grouped into four subfamilies: CXC, CC, (X)C, and CX3C.

As used herein, the term “collagen” refers to the predominant structuralprotein located within the extracellular space, and maintains themechanical integrity of many different tissues. Collagen's molecularorganization determines its function. There are more than 20 types ofcollagen currently identified, with type I being the most common.

As used herein, the term “collagen-binding domain” refers to apolypeptide, or a portion thereof, that binds to collagen. Acollagen-binding domain may be part of a larger fusion protein,bioactive agent, or pharmaceutical agent. The binding of a composition,polypeptide or portion thereof, fusion protein, or pharmaceutical orbioactive agent to collagen can determined by methods known in the art(e.g., collagen-binding assay; see e.g., Turecek et al., (2002) SeminThromb Hemost 28(2): 149-160). In some embodiments, a collagen-bindingdomain is determined by its ability to compete with a known or referencecollagen-binding protein for binding to collagen. In some embodiments, acollagen-binding domains is derived from a naturally-occurringcollagen-binding protein or collagen receptor. Collagen-binding proteinsand collagen receptors comprising collagen-binding domains are known inthe art (see e.g., Svensson et al., (2001) Osteoarthritis Cartilage 9Suppl A:S23-28; Leitinger and Hohenester E (2007) Matrix Biol26(3):146-155). In some embodiments, the collagen-binding domain isderived from a prokaryotic collagen-binding protein. Prokaryoticcollagen-binding proteins are known in the art (see e.g., Symersky etal., (1997) Nat Struct Biol 4:833-838). In some embodiments, acollagen-binding domain comprises one or more mutations that increasesits affinity for collagen.

As used herein, “combination therapy” embraces administration of eachagent or therapy in a sequential or simultaneous manner in a regimenthat will provide beneficial effects of the combination, andco-administration of these agents or therapies in a substantiallysimultaneous manner, such as in a single capsule having a fixed ratio ofthese active agents or in multiple, separate capsules for each agent.Combination therapy also includes combinations where individual elementsmay be administered at different times and/or by different routes butwhich act in combination to provide a beneficial effect by co-action orpharmacokinetic and pharmacodynamics effect of each agent or tumortreatment approaches of the combination therapy.

A “co-stimulatory signal”, as used herein, refers to a signal, which incombination with a primary signal, such as TCR/CD3 ligation, leads to Tcell proliferation and/or upregulation or downregulation of keymolecules

“Cytotoxic T Lymphocyte Associated Antigen-4 (CTLA-4)” is a T cellsurface molecule and is a member of the immunoglobulin superfamily. Thisprotein downregulates the immune system by binding to CD80 and CD86. Theterm “CTLA-4” as used herein includes human CTLA-4 (hCTLA-4), variants,isoforms, and species homologs of hCTLA-4, and analogs having at leastone common epitope with hCTLA-4. The complete hCTLA-4 sequence can befound under GenBank Accession No. P16410 (SEQ ID NO: 46):

A polypeptide or amino acid sequence “derived from” a designatedpolypeptide or protein refers to the origin of the polypeptide.Preferably, the polypeptide or amino acid sequence which is derived froma particular sequence has an amino acid sequence that is essentiallyidentical to that sequence or a portion thereof, wherein the portionconsists of at least 10-20 amino acids, preferably at least 20-30 aminoacids, more preferably at least 30-50 amino acids, or which is otherwiseidentifiable to one of ordinary skill in the art as having its origin inthe sequence. Polypeptides derived from another peptide may have one ormore mutations relative to the starting polypeptide, e.g., one or moreamino acid residues which have been substituted with another amino acidresidue or which has one or more amino acid residue insertions ordeletions. A polypeptide can comprise an amino acid sequence which isnot naturally occurring. Such variants necessarily have less than 100%sequence identity or similarity with the starting molecule. In certainembodiments, the variant will have an amino acid sequence from about 75%to less than 100% amino acid sequence identity or similarity with theamino acid sequence of the starting polypeptide, more preferably fromabout 80% to less than 100%, more preferably from about 85% to less than100%, more preferably from about 90% to less than 100% (e.g., 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%) and most preferably from about 95% toless than 100%, e.g., over the length of the variant molecule.

In certain embodiments, there is one amino acid difference between astarting polypeptide sequence and the sequence derived therefrom.Identity or similarity with respect to this sequence is defined hereinas the percentage of amino acid residues in the candidate sequence thatare identical (i.e., same residue) with the starting amino acidresidues, after aligning the sequences and introducing gaps, ifnecessary, to achieve the maximum percent sequence identity. In certainembodiments, a polypeptide consists of, consists essentially of, orcomprises an amino acid sequence selected from the Sequence SummaryTable. In certain embodiments, a polypeptide includes an amino acidsequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an aminoacid sequence selected from the Sequence Summary Table. In certainembodiments, a polypeptide includes a contiguous amino acid sequence atleast 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a contiguous aminoacid sequence selected from the Sequence Summary Table. In certainembodiments, a polypeptide includes an amino acid sequence having atleast 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85,90, 95, 100, 200, 300, 400, or 500 (or any integer within these numbers)contiguous amino acids of an amino acid sequence selected from theSequence Summary Table.

In certain embodiments, the peptides of the disclosure are encoded by anucleotide sequence. Nucleotide sequences of the disclosure can beuseful for a number of applications, including: cloning, gene therapy,protein expression and purification, mutation introduction, DNAvaccination of a host in need thereof, antibody generation for, e.g.,passive immunization, PCR, primer and probe generation, and the like. Incertain embodiments, the nucleotide sequence of the disclosurecomprises, consists of, or consists essentially of, a nucleotidesequence selected from SEQ ID NOs: 3, 5, 7, 9, 11, 15, 17, 19, 21, 23,25, 27, 29, 31, and 33. In certain embodiments, a nucleotide sequenceincludes a nucleotide sequence at least 80%, 81%, 82%, 83%, 84%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identical to a nucleotide sequence set forth in the Sequence SummaryTable. In certain embodiments, a nucleotide sequence includes acontiguous nucleotide sequence at least 80%, 81%, 82%, 83%, 84%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identical to a contiguous nucleotide sequence set forth in the SequenceSummary Table. In certain embodiments, a nucleotide sequence includes anucleotide sequence having at least 10, 15, 20, 25, 30, 35, 40, 45, 50,55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 300, 400, or 500 (or anyinteger within these numbers) contiguous nucleotides of a nucleotidesequence set forth in the Sequence Summary Table.

It will also be understood by one of ordinary skill in the art that thepolypeptides suitable for use in the immunomodulatory fusion proteinsdisclosed herein may be altered such that they vary in sequence from thenaturally occurring or native sequences from which they were derived,while retaining the desirable activity of the native sequences. Forexample, nucleotide or amino acid substitutions leading to conservativesubstitutions or changes at “non-essential” amino acid residues may bemade. Mutations may be introduced by standard techniques, such assite-directed mutagenesis and PCR-mediated mutagenesis.

The polypeptides suitable for use in the immunomodulatory fusionproteins disclosed herein may comprise conservative amino acidsubstitutions at one or more amino acid residues, e.g., at essential ornon-essential amino acid residues. A “conservative amino acidsubstitution” is one in which the amino acid residue is replaced with anamino acid residue having a similar side chain. Families of amino acidresidues having similar side chains have been defined in the art,including basic side chains (e.g., lysine, arginine, histidine), acidicside chains (e.g., aspartic acid, glutamic acid), uncharged polar sidechains (e.g., glycine, asparagine, glutamine, serine, threonine,tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine,leucine, isoleucine, proline, phenylalanine, methionine, tryptophan),beta-branched side chains (e.g., threonine, valine, isoleucine) andaromatic side chains (e.g., tyrosine, phenylalanine, tryptophan,histidine). Thus, a nonessential amino acid residue in a bindingpolypeptide is preferably replaced with another amino acid residue fromthe same side chain family. In certain embodiments, a string of aminoacids can be replaced with a structurally similar string that differs inorder and/or composition of side chain family members. Alternatively, incertain embodiments, mutations may be introduced randomly along all orpart of a coding sequence, such as by saturation mutagenesis, and theresultant mutants can be incorporated into binding polypeptides of thedisclosure and screened for their ability to bind to the desired target.

As used herein, the term “effector cell” or “effector immune cell”refers to a cell involved in an immune response, e.g., in the promotionof an immune effector response. In some embodiments, immune effectorcells specifically recognize an antigen. Examples of immune effectorcells include, but are not limited to, Natural Killer (NK) cells, Bcells, monocytes, macrophages, T cells (e.g., cytotoxic T lymphocytes(CTLs). In some embodiments, the effector cell is a T cell. As usedherein, the term “immune effector function” or “immune effectorresponse” refers to a function or response of an immune effector cellthat promotes an immune response to a target.

As used herein, the term “Fc region” refers to the portion of a nativeimmunoglobulin formed by the respective Fc domains (or Fc moieties) ofits two heavy chains. In some embodiments, the term “Fc domain” refersto a portion of a single immunoglobulin (Ig) heavy chain wherein the Fcdomain does not comprise an Fv domain. In some embodiments, the term “Fcdomain” refers to a portion of a single immunoglobulin (Ig) heavy chainalso comprising an Fv domain. As such, an Fc domain can also be referredto as “Ig” or “IgG.” In certain embodiments, an Fc domain begins in thehinge region just upstream of the papain cleavage site and ends at theC-terminus of the antibody. Accordingly, a complete Fc domain comprisesat least a hinge domain, a CH2 domain, and a CH3 domain. In certainembodiments, an Fc domain comprises at least one of: a hinge (e.g.,upper, middle, and/or lower hinge region) domain, a CH2 domain, a CH3domain, a CH4 domain, or a variant, portion, or fragment thereof. Incertain embodiments, an Fc domain comprises a complete Fc domain (i.e.,a hinge domain, a CH2 domain, and a CH3 domain). In certain embodiments,an Fc domain comprises a hinge domain (or portion thereof) fused to aCH3 domain (or portion thereof). In certain embodiments, an Fc domaincomprises a CH2 domain (or portion thereof) fused to a CH3 domain (orportion thereof). In certain embodiments, an Fc domain consists of a CH3domain or portion thereof. In certain embodiments, an Fc domain consistsof a hinge domain (or portion thereof) and a CH3 domain (or portionthereof). In certain embodiments, an Fc domain consists of a CH2 domain(or portion thereof) and a CH3 domain. In certain embodiments, an Fcdomain consists of a hinge domain (or portion thereof) and a CH2 domain(or portion thereof). In certain embodiments, an Fc domain lacks atleast a portion of a CH2 domain (e.g., all or part of a CH2 domain). AnFc domain herein generally refers to a polypeptide comprising all orpart of the Fc domain of an immunoglobulin heavy-chain. This includes,but is not limited to, polypeptides comprising the entire CH1, hinge,CH2, and/or CH3 domains as well as fragments of such peptides comprisingonly, e.g., the hinge, CH2, and CH3 domain. The Fc domain may be derivedfrom an immunoglobulin of any species and/or any subtype, including, butnot limited to, a human IgG1, IgG2, IgG3, IgG4, IgD, IgA, IgE, or IgMantibody. A human IgG1 constant region can be found at Uniprot P01857and SEQ ID NO: 114. The Fc domain of human IgG1 can be found in SEQ IDNO: 115. The Fc domain encompasses native Fc and Fc variant molecules.As with Fc variants and native Fc's, the term Fc domain includesmolecules in monomeric or multimeric form, whether digested from wholeantibody or produced by other means. The assignment of amino acidresidue numbers to an Fc domain is in accordance with the definitions ofKabat. See, e.g., Sequences of Proteins of Immunological Interest (Tableof Contents, Introduction and Constant Region Sequences sections), 5thedition, Bethesda, Md.:NIH vol. 1:647-723 (1991); Kabat et al.,“Introduction” Sequences of Proteins of Immunological Interest, US Deptof Health and Human Services, NIH, 5th edition, Bethesda, Md. vol.l:xiii-xcvi (1991); Chothia & Lesk, J. Mol. Biol. 196:901-917 (1987);Chothia et al., Nature 342:878-883 (1989), each of which is hereinincorporated by reference for all purposes.

As set forth herein, it will be understood by one of ordinary skill inthe art that any Fc domain may be modified such that it varies in aminoacid sequence from the native Fc domain of a naturally occurringimmunoglobulin molecule. In certain embodiments, the Fc domain hasreduced effector function (e.g., FcγR binding).

The Fc domains suitable for use in the immunomodulatory fusion proteinsdisclosed herein may be derived from different immunoglobulin molecules.For example, an Fc domain of a polypeptide may comprise a CH2 and/or CH3domain derived from an IgG1 molecule and a hinge region derived from anIgG3 molecule. In another example, an Fc domain can comprise a chimerichinge region derived, in part, from an IgG1 molecule and, in part, froman IgG3 molecule. In another example, an Fc domain can comprise achimeric hinge derived, in part, from an IgG1 molecule and, in part,from an IgG4 molecule.

As used herein, the term “gly-ser polypeptide linker” or “gly-serlinker” refers to a peptide that consists of glycine and serineresidues. An exemplary gly-ser polypeptide linker comprises the aminoacid sequence Ser(Gly₄Ser)n. In certain embodiments, n=1. In certainembodiments, n=2. In certain embodiments, n=3, i.e., Ser(Gly₄Ser)3. Incertain embodiments, n=4, i.e., Ser(Gly₄Ser)4. In certain embodiments,n=5. In certain embodiments, n=6. In certain embodiments, n=7. Incertain embodiments, n=8. In certain embodiments, n=9. In certainembodiments, n=10. Another exemplary gly-ser polypeptide linkercomprises the amino acid sequence (Gly₄Ser)n. In certain embodiments,n=1. In certain embodiments, n=2. In certain embodiments, n=3. Incertain embodiments, n=4. In certain embodiments, n=5. In certainembodiments, n=6. Another exemplary gly-ser polypeptide linker comprisesthe amino acid sequence (Gly₃Ser)n. certain embodiments, n=1. In certainembodiments, n=2. In certain embodiments, n=3. In certain embodiments,n=4. In certain embodiments, n=5. In certain embodiments, n=6.

As used herein, the term “human antibody” includes antibodies havingvariable and constant regions (if present) of human germlineimmunoglobulin sequences. Human antibodies of the disclosure can includeamino acid residues not encoded by human germline immunoglobulinsequences (e.g., mutations introduced by random or site-specificmutagenesis in vitro or by somatic mutation in vivo) (see, Lonberg, N.et al. (1994) Nature 368(6474): 856-859); Lonberg, N. (1994) Handbook ofExperimental Pharmacology 113:49-101; Lonberg, N. and Huszar, D. (1995)Intern. Rev. Immunol. Vol. 13: 65-93, and Harding, F. and Lonberg, N.(1995) Ann. N.Y. Acad. Sci 764:536-546). However, the term “humanantibody” does not include antibodies in which CDR sequences derivedfrom the germline of another mammalian species, such as a mouse, havebeen grafted onto human framework sequences (i.e., humanizedantibodies).

As used herein, the term a “heterologous antibody” is defined inrelation to the transgenic non-human organism producing such anantibody. This term refers to an antibody having an amino acid sequenceor an encoding nucleic acid sequence corresponding to that found in anorganism not consisting of the transgenic non-human animal, andgenerally from a species other than that of the transgenic non-humananimal.

As used herein, “immune cell” is a cell of hematopoietic origin and thatplays a role in the immune response. Immune cells include lymphocytes(e.g., B cells and T cells), natural killer cells, and myeloid cells(e.g., monocytes, macrophages, eosinophils, mast cells, basophils, andgranulocytes).

As used herein, “immune checkpoint” refers to co-stimulatory andinhibitory signals that regulates immune cells\. In certain embodiments,the immune checkpoint is an inhibitory signal. In certain embodiments,the inhibitory signal is the interaction between PD-1 and PD-L1. Incertain embodiments, the inhibitory signal is the interaction betweenCTLA-4 and CD80 or CD86 to displace CD28 binding. In certain embodimentsthe inhibitory signal is the interaction between LAG3 and MHC class IImolecules. In certain embodiments, the inhibitory signal is theinteraction between TIM3 and galectin 9.

As used herein, “immune checkpoint blocker” refers to a molecule thattotally or partially reduces, inhibits, interferes with or modulates oneor more checkpoint proteins. In certain embodiments, the immunecheckpoint blocker prevents inhibitory signals associated with theimmune checkpoint. In certain embodiments, the immune checkpoint blockeris an antibody, or fragment thereof that disrupts inhibitory signalingassociated with the immune checkpoint. In certain embodiments, theimmune checkpoint blocker is a small molecule that disrupts inhibitorysignaling. In certain embodiments, the immune checkpoint blocker is anantibody, fragment thereof, or antibody mimic, that prevents theinteraction between checkpoint blocker proteins, e.g., an antibody, orfragment thereof, that prevents the interaction between PD-1 and PD-L1.In certain embodiments, the immune checkpoint blocker is an antibody, orfragment thereof, that prevents the interaction between CTLA-4 and CD80or CD86. In certain embodiments, the immune checkpoint blocker is anantibody, or fragment thereof, that prevents the interaction betweenLAG3 and its ligands, or TIM-3 and its ligands.

As used herein, the term “immunomodulatory fusion protein” refers to apolypeptide comprising a collagen-binding domain operably linked to atleast one immunomodulatory domain. In some embodiments, thecollagen-binding domain is operably linked to the immunomodulatorydomain via a linker.

As used herein, the term “immunomodulatory domain” refers to apolypeptide (e.g., cytokine, agonist or antagonistic antibody) thatconfers an activity resulting in activation or suppression of an immuneresponse (e.g., stimulation of CD8+ T cells). In some embodiments, theimmunomodulatory domain refers to a polypeptide that binds to itscognate ligand or receptor, thereby resulting in activation orsuppression of an immune response.

The terms “inducing an immune response” and “enhancing an immuneresponse” are used interchangeably and refer to the stimulation of animmune response (i.e., either passive or adaptive) to a particularantigen. The term “induce” as used with respect to inducing CDC or ADCCrefer to the stimulation of particular direct cell killing mechanisms.

As used herein, a subject “in need of prevention,” “in need oftreatment,” or “in need thereof,” refers to one, who by the judgment ofan appropriate medical practitioner (e.g., a doctor, a nurse, or a nursepractitioner in the case of humans; a veterinarian in the case ofnon-human mammals), would reasonably benefit from a given treatment(such as treatment with a composition comprising a fusion proteindescribed herein).

The term “in vivo” refers to processes that occur in a living organism.

As used herein, “interleukin (IL)-2,” refers to a pleiotropic cytokinethat activates and induces proliferation of T cells and natural killer(NK) cells. IL-2 signals by binding its receptor, IL-2R, which iscomprised of alpha, beta, and gamma subunits. IL-2 signaling stimulatesproliferation of antigen-activated T cells.

As used herein, the term “isolated antibody” is intended to refer to anantibody which is substantially free of other antibodies havingdifferent antigenic specificities (e.g., an isolated antibody that bindsto immune checkpoint blockers or co-stimulatory molecules) issubstantially free of antibodies that specifically bind antigens otherthan the target of interest. An isolated antibody that specificallybinds to an epitope may, however, have cross-reactivity to other targetsfrom different species. In addition, an isolated antibody is typicallysubstantially free of other cellular material and/or chemicals.

As used herein, the term “isolated nucleic acid molecule” refers tonucleic acids encoding fusion proteins, polypeptides, antibodies orantibody portions disclosed herein, is intended to refer to a nucleicacid molecule in which the nucleotide sequences encoding the fusionprotein, polypeptide, antibody or antibody portion are free of othernucleotide sequences, which other sequences may naturally flank thenucleic acid in human genomic DNA.

As used herein, “isotype” refers to the antibody class (e.g., IgM orIgG1) that is encoded by heavy chain constant region genes. In someembodiments, an antibody of the disclosure is of the IgG1 isotype. Insome embodiments, an antibody of the disclosure is of the IgG2 isotype.In some embodiments, an antibody of the disclosure is of the IgG3isotype. In some embodiments, an antibody of the disclosure is of theIgG4 isotype.

As used herein the term “KD” or “K_(D)” refers to the equilibriumdissociation constant of a binding reaction between e.g., a ligand and areceptor, an antigen and an antibody, or a collagen-binding protein andcollagen. The value of K_(D) is a numeric representation of the ratio ofthe binding protein off-rate constant (kd) to the binding proteinon-rate constant (ka). The value of K_(D) is inversely related to thebinding affinity of the binding protein to its binding partner. Thesmaller the K_(D) value the greater the affinity of the binding proteinfor its binding partner. Affinity is the strength of binding of a singlemolecule to its ligand and is typically measured and reported by theequilibrium dissociation constant (K_(D)), which is used to evaluate andrank order strengths of bimolecular interactions.

As used herein, the term “kd” or “k_(d)” (alternatively “koff” or“k_(off)”) is intended to refer to the off-rate constant for thedissociation of a binding protein from binding protein/partner complex.The value of kd is a numeric representation of the fraction of complexesthat decay or dissociate per second, and is expressed in units sec⁻¹.

As used herein, the term “ka” or “k_(a)” (alternatively “kon” or“k_(on)”) is intended to refer to the on-rate constant for theassociation of a binding protein with a binding partner. The value of kais a numeric representation of the number of antibody/antigen complexesformed per second in a 1 molar (1M) solution of binding partners, and isexpressed in units M⁻¹ sec⁻¹.

As used herein, the terms “linked,” “operably linked,” “fused” or“fusion”, are used interchangeably. These terms refer to the joiningtogether of two more elements or components or domains, by whatevermeans including chemical conjugation, noncovalent complex formation orrecombinant means. Methods of chemical conjugation (e.g., usingheterobifunctional crosslinking agents) are known in the art.

As used herein, “local administration” or “local delivery,” refers todelivery that does not rely upon transport of the composition or agentto its intended target tissue or site via the vascular system. Forexample, the immunomodulatory fusion protein or composition comprisingthe fusion protein may be delivered by injection or implantation of thefusion protein or composition, or by injection or implantation of adevice containing the fusion protein or composition. Following localadministration in the vicinity of a target tissue or site, thecomposition or agent, or one or more components thereof, may diffuse tothe intended target tissue or site. In some embodiments, animmunomodulatory fusion protein is locally administered by viralvectors, electroporation, transplantation of cells expressing theimmunomodulatory fusion protein, or replicons.

“Lymphocyte Activation Gene-3 (LAG3)” is an inhibitory receptorassociated with inhibition of lymphocyte activity by binding to MHCclass II molecules. This receptor enhances the function of Treg cellsand inhibits CD8+ effector T cell function. The term “LAG3” as usedherein includes human LAG3 (hLAG3), variants, isoforms, and specieshomologs of hLAG3, and analogs having at least one common epitope. Thecomplete hLAG3 sequence can be found under GenBank Accession No. P18627(SEQ ID NO: 47).

The term “mammal” or “subject” or “patient” as used herein includes bothhumans and non-humans and includes, but is not limited to, humans,non-human primates, canines, felines, murines, bovines, equines, andporcines.

“Nucleic acid” refers to deoxyribonucleotides or ribonucleotides andpolymers thereof in either single- or double-stranded form. Unlessspecifically limited, the term encompasses nucleic acids containingknown analogues of natural nucleotides that have similar bindingproperties as the reference nucleic acid and are metabolized in a mannersimilar to naturally occurring nucleotides. Unless otherwise indicated,a particular nucleic acid sequence also implicitly encompassesconservatively modified variants thereof (e.g., degenerate codonsubstitutions) and complementary sequences and as well as the sequenceexplicitly indicated. Specifically, degenerate codon substitutions canbe achieved by generating sequences in which the third position of oneor more selected (or all) codons is substituted with mixed-base and/ordeoxyinosine residues (Batzer et al., Nucleic Acid Res. 19:5081, 1991;Ohtsuka et al., Biol. Chem. 260:2605-2608, 1985; and Cassol et al, 1992;Rossolini et al, Mol. Cell. Probes 8:91-98, 1994). For arginine andleucine, modifications at the second base can also be conservative. Theterm nucleic acid is used interchangeably with gene, cDNA, and mRNAencoded by a gene.

Polynucleotides used herein can be composed of any polyribonucleotide orpolydeoxribonucleotide, which can be unmodified RNA or DNA or modifiedRNA or DNA. For example, polynucleotides can be composed of single- anddouble-stranded DNA, DNA that is a mixture of single- anddouble-stranded regions, single- and double-stranded RNA, and RNA thatis mixture of single- and double-stranded regions, hybrid moleculescomprising DNA and RNA that can be single-stranded or, more typically,double-stranded or a mixture of single- and double-stranded regions. Inaddition, the polynucleotide can be composed of triple-stranded regionscomprising RNA or DNA or both RNA and DNA. A polynucleotide can alsocontain one or more modified bases or DNA or RNA backbones modified forstability or for other reasons. “Modified” bases include, for example,tritylated bases and unusual bases such as inosine. A variety ofmodifications can be made to DNA and RNA; thus, “polynucleotide”embraces chemically, enzymatically, or metabolically modified forms.

As used herein, “parenteral administration,” “administeredparenterally,” and other grammatically equivalent phrases, refer tomodes of administration other than enteral and topical administration,usually by injection, and include, without limitation, intravenous,intranasal, intraocular, intramuscular, intraarterial, intrathecal,intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal,transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular,subarachnoid, intraspinal, epidural, intracerebral, intracranial,intracarotid, intracolonic/intraintestinal, intravervical/intravaginal,and intrasternal injection and infusion.

The term “percent identity,” in the context of two or more nucleic acidor polypeptide sequences, refer to two or more sequences or subsequencesthat have a specified percentage of nucleotides or amino acid residuesthat are the same, when compared and aligned for maximum correspondence,as measured using one of the sequence comparison algorithms describedbelow (e.g., BLASTP and BLASTN or other algorithms available to personsof skill) or by visual inspection. Depending on the application, the“percent identity” can exist over a region of the sequence beingcompared, e.g., over a functional domain, or, alternatively, exist overthe full length of the two sequences to be compared. For sequencecomparison, typically one sequence acts as a reference sequence to whichtest sequences are compared. When using a sequence comparison algorithm,test and reference sequences are input into a computer, subsequencecoordinates are designated, if necessary, and sequence algorithm programparameters are designated. The sequence comparison algorithm thencalculates the percent sequence identity for the test sequence(s)relative to the reference sequence, based on the designated programparameters.

Optimal alignment of sequences for comparison can be conducted, e.g., bythe local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482(1981), by the homology alignment algorithm of Needleman & Wunsch, J.Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson& Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444 (1988), by computerizedimplementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA inthe Wisconsin Genetics Software Package, Genetics Computer Group, 575Science Dr., Madison, Wis.), or by visual inspection (see generallyAusubel et al., infra).

One example of an algorithm that is suitable for determining percentsequence identity and sequence similarity is the BLAST algorithm, whichis described in Altschul et al., J. Mol. Biol. 215:403-410 (1990).Software for performing BLAST analyses is publicly available through theNational Center for Biotechnology Information website.

As generally used herein, “pharmaceutically acceptable” refers to thosecompounds, materials, compositions, and/or dosage forms which are,within the scope of sound medical judgment, suitable for use in contactwith the tissues, organs, and/or bodily fluids of human beings andanimals without excessive toxicity, irritation, allergic response, orother problems or complications commensurate with a reasonablebenefit/risk ratio.

“Polypeptide,” “peptide”, and “protein” are used interchangeably hereinto refer to a polymer of amino acid residues. The terms apply to aminoacid polymers in which one or more amino acid residue is an artificialchemical mimetic of a corresponding naturally occurring amino acid, aswell as to naturally occurring amino acid polymers and non-naturallyoccurring amino acid polymer.

The “Programmed Death-1 (PD-1)” receptor refers to an immuno-inhibitoryreceptor belonging to the CD28 family. PD-1 is expressed predominantlyon previously activated T cells in vivo, and binds to two ligands, PD-L1and PD-L2. The term “PD-1” as used herein includes human PD-1 (hPD-1),variants, isoforms, and species homologs of hPD-1, and analogs having atleast one common epitope with hPD-1. The complete hPD-1 sequence can befound under GenBank Accession No. AAC51773 (SEQ ID NO: 44).

“Programmed Death Ligand-1 (PD-L1)” is one of two cell surfaceglycoprotein ligands for PD-1 (the other being PD-L2) that downregulatesT cell activation and cytokine secretion upon binding to PD-1. The term“PD-L1” as used herein includes human PD-L1 (hPD-L1), variants,isoforms, and species homologs of hPD-L1, and analogs having at leastone common epitope with hPD-L1. The complete hPD-L1 sequence can befound under GenBank Accession No. Q9NZQ7 (SEQ ID NO: 45).

As used herein, the term “purified” or “isolated” as applied to any ofthe proteins (fusion proteins, antibodies or fragments) described hereinrefers to a polypeptide that has been separated or purified fromcomponents (e.g., proteins or other naturally-occurring biological ororganic molecules) which naturally accompany it, e.g., other proteins,lipids, and nucleic acid in a prokaryote expressing the proteins.Typically, a polypeptide is purified when it constitutes at least 60(e.g., at least 65, 70, 75, 80, 85, 90, 92, 95, 97, or 99) %, by weight,of the total protein in a sample.

As used herein, the terms “specifically binds” and “selectively binds”refers to binding by a collagen-binding domain to collagen, or bindingby an antibody to an epitope on a predetermined antigen. In someembodiments, a collagen-binding domain specifically binds or selectivelybinds to collagen based on the K_(D) for collagen (i.e., the K_(D) forbinding to collagen is lower than the K_(D) for at least fibronectin,vitronectin, osteopontin, tenascin C or fibrinogen).

The term “sufficient amount” or “amount sufficient to” means an amountsufficient to produce a desired effect, e.g., an amount sufficient toreduce the size of a tumor.

The term “T cell” refers to a type of white blood cell that can bedistinguished from other white blood cells by the presence of a T cellreceptor on the cell surface. There are several subsets of T cells,including, but not limited to, T helper cells (a.k.a. T_(H) cells orCD4⁺ T cells) and subtypes, including T_(H)1, T_(H)2, T_(H)3, T_(H)17,T_(H)9, and T_(FH) cells, cytotoxic T cells (a.k.a T_(C) cells, CD8⁺ Tcells, cytotoxic T lymphocytes, T-killer cells, killer T cells), memoryT cells and subtypes, including central memory T cells (T_(CM) cells),effector memory T cells (T_(EM) and T_(EMRA) cells), and resident memoryT cells (T_(RM) cells), regulatory T cells (a.k.a. T_(reg) cells orsuppressor T cells) and subtypes, including CD4⁺ FOXP3⁺ T_(reg) cells,CD4+FOXP3⁻ T_(reg) cells, Tr1 cells, Th3 cells, and T_(reg)17 cells,natural killer T cells (a.k.a. NKT cells), mucosal associated invariantT cells (MAITs), and gamma delta T cells (γδ T cells), including Vγ9/Vδ2T cells. Any one or more of the aforementioned or unmentioned T cellsmay be the target cell type for a method as disclosed herein.

The term “T cell cytotoxicity” includes any immune response that ismediated by CD8+ T cell activation. Exemplary immune responses includecytokine production, CD8+ T cell proliferation, granzyme or perforinproduction, and clearance of an infectious agent.

“T Cell Membrane Protein-3 (TIM3)” is an inhibitory receptor involved inthe inhibition of lymphocyte activity by inhibition of T_(H)1 cellsresponses. Its ligand is galectin 9, which is upregulated in varioustypes of cancers. The term “TIM3” as used herein includes human TIM3(hTIM3), variants, isoforms, and species homologs of hTIM3, and analogshaving at least one common epitope. The complete hTIM3 sequence can befound under GenBank Accession No. Q8TDQo (SEQ ID NO: 48).

A “therapeutic antibody” is an antibody, fragment of an antibody, orconstruct that is derived from an antibody, and can bind to acell-surface antigen on a target cell to cause a therapeutic effect.Such antibodies can be chimeric, humanized or fully human antibodies.Methods are known in the art for producing such antibodies. Suchantibodies include single chain Fc fragments of antibodies, minibodiesand diabodies. Any of the therapeutic antibodies known in the art to beuseful for cancer therapy can be used in the combination therapysuitable for use in the methods disclosed herein. Therapeutic antibodiesmay be monoclonal antibodies or polyclonal antibodies. In preferredembodiments, the therapeutic antibodies target cancer antigens.

The term “therapeutically effective amount” is an amount that iseffective to ameliorate a symptom of a disease. A therapeuticallyeffective amount can be a “prophylactically effective amount” asprophylaxis can be considered therapy.

As used herein, the term “vector” is intended to refer to a nucleic acidmolecule capable of transporting another nucleic acid to which it hasbeen linked. One type of vector is a “plasmid,” which refers to acircular double stranded DNA loop into which additional DNA segments maybe ligated. Another type of vector is a viral vector, wherein additionalDNA segments may be ligated into the viral genome. Certain vectors arecapable of autonomous replication in a host cell into which they areintroduced (e.g., bacterial vectors having a bacterial origin ofreplication and episomal mammalian vectors). Other vectors (e.g.,non-episomal mammalian vectors) can be integrated into the genome of ahost cell upon introduction into the host cell, and thereby arereplicated along with the host genome. Moreover, certain vectors arecapable of directing the expression of genes to which they areoperatively linked. Such vectors are referred to herein as “recombinantexpression vectors” (or simply, “expression vectors”). In general,expression vectors of utility in recombinant DNA techniques are often inthe form of plasmids. In the present specification, “plasmid” and“vector” may be used interchangeably as the plasmid is the most commonlyused form of vector. However, the disclosure is intended to include suchother forms of expression vectors, such as viral vectors (e.g.,replication defective retroviruses, adenoviruses and adeno-associatedviruses), which serve equivalent functions.

Immunomodulatory Fusion Protein

In some aspects, the disclosure provides an immunomodulatory fusionprotein comprising a collagen-binding domain operably linked to animmunomodulatory domain. In some embodiments, the immunomodulatoryfusion protein further comprises a linker, such that thecollagen-binding domain is operably linked to a linker, and the linkeris operably linked to the immunomodulatory domain.

I. Collagen-Binding Domains

In some embodiments, the disclosure provides immunomodulatory fusionproteins comprising a collagen-binding domain. In some embodiments, thecollagen-binding domain has a MW of about 5-100 kDa, about 10-80 kDa,about 20-60 kDa, about 30-50 kDa, or about 10 kDa, about 20 kDa, about30 kDa, about 40 kDa, about 50 kDa, about 60 kDa, about 70 kDa, about 80kDa, about 90 kDa or about 100 kDa. In some embodiments, thecollagen-binding domain is about 5 kDa, about 10 kDa, about 20 kDa,about 30 kDa, about 40 kDa, about 50 kDa, about 60 kDa, about 70 kDa,about 80 kDa, about 90 kDa, or about 100 kDa. In some embodiments, thecollagen-binding domain is about 30 kDa. In some embodiments, thecollagen-binding domain is about 40 kDa.

In some embodiments, the collagen-binding domain is about 10-350, about10-300, about 10-250, about 10-200, about 10-150, about 10-100, about10-50, or about 10-20 amino acids in length. In some embodiments, thecollagen-binding domain is about 10 amino acids in length. In someembodiments, the collagen-binding domain is about 15 amino acids inlength. In some embodiments, the collagen-binding domain is about 20amino acids in length. In some embodiments, the collagen-binding domainis about 30 amino acids in length. In some embodiments, thecollagen-binding domain is about 40 amino acids in length. In someembodiments, the collagen-binding domain is about 50 amino acids inlength. In some embodiments, the collagen-binding domain is about 60amino acids in length. In some embodiments, the collagen-binding domainis about 70 amino acids in length. In some embodiments, thecollagen-binding domain is about 80 amino acids in length. In someembodiments, the collagen-binding domain is about 90 amino acids inlength. In some embodiments, the collagen-binding domain is about 100amino acids in length. In some embodiments, the collagen-binding domainis about 120 amino acids in length. In some embodiments, thecollagen-binding domain is about 150 amino acids in length. In someembodiments, the collagen-binding domain is about 200 amino acids inlength. In some embodiments, the collagen-binding domain is about 250amino acids in length. In some embodiments, the collagen-binding domainis about 300 amino acids in length. In some embodiments, thecollagen-binding domain is about 350 amino acids in length.

A. Isoelectric Point

The isoelectric point (pI, pH(I), IEP), is the pH at which a particularmolecule (e.g., a collagen-binding domain) carries no net electricalcharge or is electrically neutral. Table 1 provides the calculated pIfor exemplary collagen-binding domains, described herein. The ExPASytool from Swiss Institute of Bioinformatics(https://web.expasy.org/compute_pi/) was used to calculate theisoelectric points (pI) of the collagen-binding domains shown in Table1.

In some embodiments, the collagen-binding domain has an isoelectricpoint pI less than (<) about 10, about 8, about 6, about 4, about 2, orabout 1. In some embodiments, the collagen-binding domain has anisoelectric point pI of less than (<) 10. In some embodiments, thecollagen-binding domain has an isoelectric point pI of less than (<) 10and a molecular weight (MW) of greater than (>) 5 kDa.

TABLE 1 Calculated pI for Exemplary Collagen-Binding DomainsCollagen-Binding Domain Calculated pI SEQ ID NO LAIR1 5.23 98 LAIR2 4.8899 Glycoprotein IV 7.68 100 Nidogen 5.05 101 Perlecan 6.03 102 Biglycan8.13 103 Decorin 8.76 104 Asporin 6.1 105 Fibromodulin 5.66 106 Lumican6.17 107 PRELP 9.45 108 Osteoadherin/Osteomodulin 5.22 109 Opticin 5.38110 Osteoglycin/Mimecan 5.22 111 Chondroadherin 9.14 112 Podcan 6.41 113Lumican (murine) 6.01 195

B. Type I Collagen

Collagen is the predominant structural protein located within theextracellular space and type I collagen is the most abundant protein inmammals (Di Lullo et al., (2002) J Biol Chem 277(6):4223-4231). Thefundamental structural unit of type I collagen is a long (300-nm), thin(1.5-nm-diameter) protein that consists of three coiled subunits: twoα1(I) chains and one α2(I). Each chain contains 1050 amino acids woundaround one another in a characteristic right-handed triple helix. Inhumans, type I collagen is encoded by the COL1A1 and COL1A2 genes. TheCOL1A1 gene encodes the pro-alpha1 chain of type I collagen. The COL1A2gene pro-alpha2 chain of type I collagen, whose triple helix comprisestwo alpha1 chains and one alpha2 chain. Type I is a fibril-formingcollagen found in most connective tissues and is abundant in bone,cornea, dermis and tendon.

An exemplary amino acid sequence for the human alpha1 chain precursor oftype I collagen is set forth in SEQ ID NO: 90 (NCBI Reference Sequence:NP_000079.2).

An exemplary amino acid sequence for the human alpha2 chain precursor oftype I collagen is set forth in SEQ ID NO: 91 (NCBI Reference Sequence:NP_000080.2).

C. Type IV Collagen

Type IV collagen is comprised of a family of polypeptides and is a majorconstituent of mammalian basement membranes (Timpl (1989) Eur J Biochem180:487-502; Paulsson (1992) Crit Rev Biochem Mol Biol 27:93-127). Theα1(IV) and α2(IV) chains are products of distinct genes (COL4A1 andCOL4A2, respectively) located pairwise in a head-to-head fashion onchromosome 13 in humans (Hudson et al., (1993) J Biol Chem268:26033-26036). The α3(IV) and α4(IV) chains (encoded by the COL4A3and COL4A4 genes, respectively) are present in the same orientation onchromosome 2 in humans, and the α5(IV) and α6(IV) chains (encoded by theCOL4A5 and COL4A6 genes, respectively) are located on the X chromosomein humans (Hudson et al., (1991) in Pathobiochemistry, ed Kang A. (CRCPress, Boca Raton, Fla.), pp 17-30).

An exemplary amino acid sequence for the human alpha1 chain of type IVcollagen is set forth in SEQ ID NO: 92 (NCBI Reference Sequence:XP_011519350.1).

An exemplary amino acid sequence for the human alpha2 chain of type IVcollagen is set forth in SEQ ID NO: 93 (NCBI Reference Sequence:NP_001837.2).

An exemplary amino acid sequence for the human alpha3 chain of type IVcollagen is set forth in SEQ ID NO: 94 (NCBI Reference Sequence:NP_000082.2).

An exemplary amino acid sequence for the human alpha4 chain of type IVcollagen is set forth in SEQ ID NO: 95 (NCBI Reference Sequence:NP_000083.3).

An exemplary amino acid sequence for the human alpha5 chain of type IVcollagen is set forth in SEQ ID NO: 96 (NCBI Reference Sequence:XP_011529151.2).

An exemplary amino acid sequence for the human alpha6 chain of type IVcollagen is set forth in SEQ ID NO: 97 (NCBI Reference Sequence:XP_006724680.1).

Accordingly, in some embodiments, the disclosure providesimmunomodulatory fusion proteins comprising a collagen-binding domainthat specifically binds collagen. In some embodiments, thecollagen-binding domain specifically binds human type I collagen and/orhuman type IV collagen. In some embodiments, the collagen-binding domainbinds human type I collagen. In some embodiments, the collagen-bindingdomain binds human type IV collagen. In some embodiments, thecollagen-binding domain specifically binds human type I collagen andhuman type IV collagen. In some embodiments, the collagen-binding domainspecifically binds human type I collagen or human type IV collagen.

D. Binding Affinity to Collagen

In some embodiments, the disclosure provide immunomodulatory fusionproteins comprising a collagen-binding domain that specifically bindscollagen with an affinity (K_(D)) of less than about 0.5 nM asdetermined by a collagen-binding assay. In some embodiments, thedisclosure provide immunomodulatory fusion proteins comprising acollagen-binding domain that specifically binds collagen with anaffinity (K_(D)) of less than about 5 nM as determined by acollagen-binding assay. In some embodiments, the disclosure provideimmunomodulatory fusion proteins comprising a collagen-binding domainthat specifically binds collagen with an affinity (K_(D)) of less thanabout 50 nM as determined by a collagen-binding assay. In someembodiments, the disclosure provide immunomodulatory fusion proteinscomprising a collagen-binding domain that specifically binds collagenwith an affinity (K_(D)) of less than about 500 nM as determined by acollagen-binding assay. In some embodiments, the collagen-binding domainspecifically binds collagen with an affinity (K_(D)) of about 0.5-5 nM,5-50 nM, or 50-500 nM as determined by a collagen-binding assay. In someembodiments, the collagen-binding domain specifically binds collagenwith an affinity (K_(D)) of about 50-100 nM, 100-200 nM, 200-300 nM,300-400 nM, or 400-500 nM as determined by a collagen-binding assay.

In some embodiments, the collagen-binding assay determines a bindingaffinity of the collagen-binding domain for collagen. In someembodiments, the collagen-binding assay determines a binding affinity ofthe collagen-binding domain for type I collagen. In some embodiments,the collagen-binding assay determines a binding affinity for type IVcollagen.

In some embodiments, the collagen-binding assay is an ELISA. Methods andtechniques to perform a collagen-binding ELISA are known in the art (seee.g., Smith et al., (2000) J Biol Chem 275:4205-4209). Accordingly, insome embodiments, the disclosure provides an immunomodulatory fusionprotein comprising a collagen-binding domain that specifically bindscollagen with an affinity (K_(D)) of less than about 0.5 nM asdetermined by an ELISA. Accordingly, in some embodiments, the disclosureprovides an immunomodulatory fusion protein comprising acollagen-binding domain that specifically binds collagen with anaffinity (K_(D)) of less than about 5 nM as determined by an ELISA.Accordingly, in some embodiments, the disclosure provides animmunomodulatory fusion protein comprising a collagen-binding domainthat specifically binds collagen with an affinity (K_(D)) of less thanabout 50 nM as determined by an ELISA. Accordingly, in some embodiments,the disclosure provides an immunomodulatory fusion protein comprising acollagen-binding domain that specifically binds collagen with anaffinity (K_(D)) of less than about 500 nM as determined by an ELISA. Insome embodiments, the collagen-binding domain specifically bindscollagen with an affinity (K_(D)) of about 0.5-5 nM, 4-40 nM, 50-500 nMas determined by an ELISA. In some embodiments, the collagen-bindingdomain specifically binds collagen with an affinity (K_(D)) of about50-100 nM, 100-200 nM, 200-300 nM, 300-400 nM, or 400-500 nM asdetermined by an ELISA.

In some embodiments, the collagen-binding assay is a surface plasmonresonance (SPR) assay. Methods and techniques to perform acollagen-binding SPR assay are known in the art (see e.g., Saenko etal., (2002) Anal Biochem 302(2):252-262). Accordingly, in someembodiments, the disclosure provides an immunomodulatory fusion proteincomprising a collagen-binding domain that specifically binds collagenwith an affinity (K_(D)) of less than about 500 nM as determined by anSPR assay. In some embodiments, the collagen-binding domain specificallybinds collagen with an affinity (K_(D)) of about 50-500 nM as determinedby an SPR assay. In some embodiments, the collagen-binding domainspecifically binds collagen with an affinity (K_(D)) of about 50-100 nM,100-200 nM, 200-300 nM, 300-400 nM, or 400-500 nM as determined by anSPR assay.

The phrase “surface plasmon resonance” includes an optical phenomenonthat allows for the analysis of real-time biospecific interactions bydetection of alterations in protein concentrations within a biosensormatrix, for example using the BIAcore system (Pharmacia Biosensor AB,Uppsala, Sweden and Piscataway, N.J.). For further descriptions, seeJinsson, U., et al. (1993) Ann. Biol. Clin. 51:19-26; Jönsson, U., etal. (1991) Biotechniques 11:620-627; Johnsson, B., et al. (1995) J. Mol.Recognit. 8:125-131; and Johnnson, B., et al. (1991) Anal. Biochem.198:268-277.

E. Binding Specificity to Collagen

In some embodiments, the disclosure provides immunomodulatory fusionproteins comprising a collagen-binding domain that specifically bindscollagen and does not specifically bind to one or more non-collagenextracellular matrix (ECM) components including, but not limited to,fibronectin, vitronectin, tenascin C, osteopontin and fibrinogen. Insome embodiments, the collagen-binding domain binds to collagen with alower K_(D) than to one or more non-collagen ECM components. In someembodiments, the K_(D) of the collagen-binding domain for type Icollagen is less than the K_(D) of the collagen-binding domain for anextracellular matrix component selected from fibronectin, vitronectin,osteopontin, tenascin C, or fibrinogen. In some embodiments, thecollagen-binding domain binds to collagen with about 10%, about 20%,about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about90%, about 99% lower K_(D) than to one or more non-collagen ECMcomponents. In some embodiments, the collagen-binding domain binds tocollagen with about 2-fold, about 3-fold, about 4-fold, about 5-fold,about 10-fold lower K_(D) than to one or more non-collagen ECMcomponents.

In some embodiments, the collagen-binding domain is not a promiscuousbinder of ECM components. In some embodiments, the collagen-bindingdomain does not comprise a heparin-binding domain. In some embodiments,the collagen-binding domain is not a growth factor or portion thereofwhich binds extracellular matrix.

In some embodiments, the collagen-binding domain binds to type Icollagen with a lower K_(D) than to type IV collagen. In someembodiments, the collagen-binding domain binds to type IV collagen witha lower K_(D) than to type I collagen.

In some embodiments, the collagen-binding domain competes with areference collagen-binding domain for binding to collagen. In someembodiments, the collagen-binding domain competes with a referencecollagen-binding domain for binding to type I collagen. In someembodiments, the collagen-binding domain competes with a referencecollagen-binding domain for binding to type IV collagen. In someembodiments, the collagen-binding domain competes with a referencecollagen-binding domain for binding to type I collagen and type IVcollagen. In some embodiments, the collagen-binding domain competes witha reference collagen-binding domain for binding to type I collagen butnot to type IV collagen. In some embodiments, the collagen-bindingdomain competes with a reference collagen-binding domain for binding totype IV collagen but not to type I collagen.

In some embodiments, the reference collagen-binding domain comprises oneor more (e.g., two, three, four, five, six, seven, eight, nine, ten ormore) leucine-rich repeats which bind collagen. In some embodiments, thereference collagen-binding domain comprises a proteoglycan. In someembodiments, the reference collagen-binding domain comprises aproteoglycan, wherein the proteoglycan is selected from the groupconsisting of: decorin, biglycan, fibromodulin, lumican, chondroadherin,asporin, PRELP, osteoadherin/osteomodulin, opticin, osteoglycin/mimecan,podocan, perlecan, nidogen. In some embodiments, the referencecollagen-binding domain is lumican. In some embodiments, the referencecollagen-binding domain comprises a class I small leucine-richproteoglycan (SLRP). SLRPs are known to bind collagen (Chen and Birk(2013) FEBS Journal 2120-2137). In some embodiments, the referencecollagen-binding domain comprises a class II SLRP. In some embodiments,the reference collagen-binding domain comprises a class III SLRP. Insome embodiments, the reference collagen-binding domain comprises aclass IV SLRP. In some embodiments, the reference collagen-bindingdomain comprises a class V SLRP. Further description of SLRP classes isdisclosed in Schaefer & Iozzo (2008) J Biol Chem 283(31):21305-21309,which is incorporated herein by reference it its entirety.

In some embodiments, the reference collagen-binding domain comprises theleukocyte-associated immunoglobulin-like receptor 1 (LAIR-1) protein. Insome embodiments, the reference collagen-binding domain comprises theleukocyte-associated immunoglobulin-like receptor 2 (LAIR-2) protein. Insome embodiments, the reference collagen-binding domain comprisesGlycoprotein IV.

F. Exemplary Collagen-Binding Domains

In some embodiments, the collagen-binding domain comprises one or more(e.g., two, three, four, five, six, seven, eight, nine, ten or more)leucine-rich repeats which bind collagen. In some embodiments, thecollagen-binding domain comprises a proteoglycan. In some embodiments,the collagen-binding domain comprises a proteoglycan, wherein theproteoglycan is selected from the group consisting of: decorin,biglycan, testican, bikunin, fibromodulin, lumican, chondroadherin,keratin, ECM2, epiphycan, asporin, PRELP, keratocan, osteoadherin,opticin, osteoglycan, nyctalopin, Tsukushi, podocan, podocan-likeprotein 1 versican, perlecan, nidogen, neurocan, aggrecan, and brevican.

In some embodiments, the collagen-binding domain comprises a class Ismall leucine-rich proteoglycan (SLRP). In some embodiments, thecollagen-binding domain comprises a class II SLRP. In some embodiments,the collagen-binding domain comprises a class III SLRP. In someembodiments, the collagen-binding domain comprises a class IV SLRP. Insome embodiments, the collagen-binding domain comprises a class V SLRP.Further description of SLRP classes is disclosed in Schaefer & Iozzo(2008) J Biol Chem 283(31):21305-21309, which is incorporated herein byreference it its entirety.

In some embodiments, the collagen-binding domain comprises one or moreleucine-rich repeats from a human proteoglycan Class II member of thesmall leucine-rich proteoglycan (SLRP) family. In some embodiments, theSLRP is selected from lumican, decorin, biglycan, fibromodulin, keratin,epiphycan, asporin and osteoglycin. In some embodiments, the SLRP islumican. In some embodiments, lumican comprises the amino acid sequenceas set forth in SEQ ID NO: 107.

Lumican

Lumican, also known as LUM, is an extracellular matrix protein that, inhumans, is encoded by the LUM gene on chromosome 12 (Chakravarti et al.,(1995) Genomics 27(3):481-488). Lumican is a proteoglycan Class IImember of the small leucine-rich proteoglycan (SLRP) family thatincludes decorin, biglycan, fibromodulin, keratocan, epiphycan, andosteoglycin (Iozzo & Schaefer (2015) Matrix Biology 42:11-55).

Like the other SLRPs, lumican has a molecular weight of about 40 kDa andhas four major intramolecular domains: 1) a signal peptide of 16 aminoacid residues, 2) a negatively-charged N-terminal domain containingsulfated tyrosine and disulfide bond(s), 3) ten tandem leucine-richrepeats allowing lumican to bind to collagen, and 4) a carboxyl terminaldomain of 50 amino acid residues containing two conserved cysteines 32residues apart. Kao et al., (2006) Experimental Eye Research 82(1):3-4).There are four N-linked sites within the leucine-rich repeat domain ofthe protein core that can be substituted with keratan sulfate. The coreprotein of lumican (like decorin and fibromodulin) is horseshoe shaped.This enables it bind to collagen molecules within a collagen fibril,thus helping keep adjacent fibrils apart Scott (1996) Biochemistry35(27): 8795-8799.

Leukocyte-Associated Immunoglobulin-Like Receptors (LAIR- and LAIR-2)Leukocyte-associated Ig-like receptor (LAIR)-1 is a collagen-receptorthat inhibits immune cell function upon collagen binding. Next toLAIR-1, the human genome encodes LAIR-2, a soluble homolog. Human (h)LAIR-1 is expressed on the majority of PBMC and thymocytes (Maasho etal., (2005) Mol Immunol 42: 1521-1530). Cross-linking of hLAIR-1 by mAbsin vitro delivers a potent inhibitory signal that is capable ofinhibiting immune cell function (4, 10-15). Collagens are known to benatural, high-affinity ligands for the LAIR molecules. Interaction ofhLAIR-1 with collagens directly inhibits immune cell activation in vitro(Meyaard et al., (1997) Immunity 7:283-290; Poggi (1998) Eur J Immunol28:2086-2091; Van der Vuurst de Vries et al., (1999) Eur J Immunol29:3160-3167; Lebbink et al., (2006) J Exp Med 203:1419-1425).

In some embodiments, the collagen-binding domain comprises a human typeI glycoprotein having an Ig-like domain, or an extracellular portionthereof which binds collagen. In some embodiments, the type Iglycoprotein competes with lumican for binding for binding to collagentype I. In some embodiments, the human type I glycoprotein is selectedfrom LAIR1, LAIR2, and Glycoprotein IV. In some embodiments, the humantype I glycoprotein is LAIR1. In some embodiments, the human type Iglycoprotein is LAIR1 and the collagen-binding domain comprises aminoacid residues 22-122 of the amino acid sequence as set forth in SEQ IDNO: 98.

In some embodiments, the collagen-binding domain is a variant of LAIR1,LAIR2, or Glycoprotein IV. In some embodiments, the LAIR1 variant, LAIR2variant, or Glycoprotein IV variant comprises one or more amino acidsubstitutions, additions or deletions (e.g., two, three, four, five,six, seven, eight, nine, ten or more) relative to the wild-type LAIR1,LAIR 2 or Glycoprotein IV protein sequence. In some embodiments, thecollagen-binding domain is a LAIR1 variant comprising one or more aminoacid substitutions, additions or deletions (e.g., two, three, four,five, six, seven, eight, nine, ten or more) relative to a LAIR1 proteincomprising the amino acid sequence of SEQ ID NO: 98. In someembodiments, the collagen-binding domain is a LAIR1 variant comprisingone or more amino acid substitutions, additions or deletions (e.g., two,three, four, five, six, seven, eight, nine, ten or more) in the LAIR1binding pocket (e.g., a LAIR1 binding site comprising one or moreresidues E61, S66, Y68, I102, W109, Y115, R59, E63, R100, E111 and Q112,and combinations thereof) (Brondijk et al., (2010) Blood 115:1364-1373).In some embodiments, the collagen-binding domain is a LAIR1 variantcomprising one or more amino acid substitutions, additions or deletions(e.g., two, three, four, five, six, seven, eight, nine, ten or more)outside the LAIR1 binding pocket.

In some embodiments, the collagen-binding domain is a LAIR1 varianthaving increased binding affinity to collagen relative to the collagenbinding affinity of a wild-type LAIR1 protein. In some embodiments, theLAIR1 variant demonstrates an increase in binding affinity to collagenrelative to the collagen binding affinity of a LAIR1 protein comprisingthe amino acid sequence of SEQ ID NO: 98. In some embodiments, the LAIR1variant having decreased binding affinity to collagen relative to thecollagen binding affinity of wild-type LAIR1 protein. In someembodiments, the LAIR1 variant demonstrates a decrease in bindingaffinity to collagen relative to the collagen binding affinity of aLAIR1 protein comprising the amino acid sequence of SEQ ID NO: 98.

Glycoprotein IV (CD36)

In some embodiments, the collagen-binding domain comprises GlycoproteinIV (GPIV). Glycoprotein IV binds many ligands including collagen (Tandon(1989) J Biol Chem 264(13): 7576-7583). A multifunctional glycoprotein,GPIV acts as receptor for a broad range of ligands, includingthrombospondin, fibronectin, collagen or amyloid-beta as well as oflipidic nature such as oxidized low-density lipoprotein (oxLDL), anionicphospholipids, long-chain fatty acids and bacterial diacylatedlipopeptides. GPIV is a protein that in humans is encoded by the CD36gene. The CD36 antigen is an integral membrane protein found on thesurface of many cell types in vertebrate animals. It imports fatty acidsinside cells and is a member of the class B scavenger receptor family ofcell surface proteins. In some embodiments, the CD36 comprises the aminoacid sequence set forth in SEQ ID NO: 100.

II. Immunomodulatory Domain

The immunomodulatory fusion proteins disclosed herein comprise at leastone immunomodulatory domain operably linked to a collagen-bindingdomain. In some embodiments, the immunomodulatory fusion proteincomprises one, two, three, four, or five immunomodulatory domains. Insome embodiments, when more than one immunomodulatory domain is presentin the fusion protein, the immunomodulatory domains are the same. Insome embodiments, when more than one immunomodulatory domain is presentin the fusion protein, the immunomodulatory domains are different. Insome embodiments, when more than one immunomodulatory domain is presentin the fusion protein, each domain is located at the N terminus of acollagen-binding domain. In some embodiments, when more than oneimmunomodulatory domain is present in the fusion protein, each domain islocated at the C terminus of a collagen-binding domain. In someembodiments, when more than one immunomodulatory domain is present inthe fusion protein, at least one domain is located at the N terminus ofa collagen-binding domain and at least one domain is located at the Cterminus of the collagen-binding domain.

In some embodiments, the immunomodulatory domain activates the activityof a cell of the immune system. For example, in some embodiments theimmunomodulatory domain is an immune response stimulatory, such as, butnot limited to, a cytokine, such as an interleukin, a chemokine, amember of the TNF family, an agonistic antibody, an immune checkpointblocker, or a combination thereof. In some embodiments, theimmunomodulatory domain enhances an immune response. In someembodiments, enhancement of an immune response includes stimulation of Tcells, stimulation of B cells, stimulation of dendritic cell responses,or a combination thereof. In some embodiments, enhancement of an immuneresponse results in cytokine production, antibody production,antigen-specific immune cell (e.g., CD8+ T cells or CD4+ T cells)production, stimulation of Type I interferon responses, or combinationsthereof.

In some embodiments, the immunomodulatory domain comprises a polypeptidethat activates, enhances or promotes a response by an immune cell. Insome embodiments, the immunomodulatory domain comprises a polypeptidethat inhibits, reduces or suppresses a response by an immune cell. Insome embodiments, the immune cell is a lymphoid cell, including but notlimited to T cells, B cells, NK cells and innate lymphoid cells. In someembodiments, the immune cell is a myeloid cell, including but notlimited to monocytes, neutrophils, macrophages, dendritic cells, mastcells and granulocytes.

In some embodiments, the response of the immune cell is cytokineproduction, antibody production, production of antigen-specific immunecells, or a combination thereof.

A. Interleukins

In some embodiments, the immunomodulatory domain suitable for use in theimmunomodulatory fusion proteins of the present disclosure, is aninterleukin (IL). Interleukins are secreted proteins that bind to theirspecific receptors and play a role in the communication amongleukocytes. Interleukins suitable for use as an immunomodulatory domainof the immunomodulatory fusion proteins include, but are not limited to:IL-2, IL-12, IL-15, IL-15 superagonist (IL-15SA), IL-21, IL-6, IL-5,IL-8, IL-7, IL-17, IL-23, IL-18, IL-1, IL-4, IL-3, IL-10, IL-13, andIL-9. In some embodiments, the interleukin suitable for use as animmunomodulatory domain comprises an amino acid sequence selected fromSEQ ID NOs: 1-5 and 9-24. In some embodiments, the immunomodulatorydomain is an IL-2 polypeptide. In some embodiments, the immunomodulatorydomain is an IL-12 polypeptide. In some embodiments, theimmunomodulatory domain is an IL-15 polypeptide. In some embodiments,the immunomodulatory domain is an IL-15SA polypeptide.

In some embodiments, the immunomodulatory domain is an interleukinpolypeptide that binds to a common gamma chain receptor. Interleukinsthat bind the common gamma chain receptor include, but are not limitedto, IL-2, IL-4, IL-7, IL-9, IL-13, IL-15, IL-15/IL-15R and IL-21.

In some embodiments, the immunomodulatory domain is a polypeptidebelonging to the IL-12 family. The IL-12 family comprises heterodimericligands comprised of an α subunit with helical structure (e.g.,IL-12p35, IL-23p19, IL-27p28) and a β subunit (e.g., IL-12p40, IL-23p40(which is identical to IL-12p40), EBI3). Exemplary members includeIL-12, IL-23, IL-27 and IL-35.

In some embodiments, the immunomodulatory domain is a polypeptidebelonging to the IL-1 superfamily. The Interleukin-1 (IL-1) familyconsists of 11 structurally related family members (IL-1 α, IL-1-β,IL-1Ra, IL-18, IL-33 and IL-1F5 to IL-1 F10), that are among the mostpotent immune system signaling molecules, acting through a group ofclosely related receptors. All IL-1 receptors have a similar mode ofactivation: upon binding of ligand to the primary receptor subunit (i.e.IL-IR1 for IL-1 α and β, IL-18R for IL-18 and ST2 for IL-33), a secondreceptor subunit is recruited (i.e. IL-1RAP for IL-1 α and β, IL-18RAPfor IL-18 and IL-1RAP for IL-33) and signaling is initiated viajuxtaposition of the receptor subunits' cytoplasmic Toll/IL-1 receptor(TIR) domains. The dimerized TIR domains provide a docking platform forthe MYD88 adaptor protein, which via recruitment of other intermediatesleads to activation of the pro-inflammatory nuclear factor-κ B (NF-κB)and mitogen-activated protein kinase (MAPK) pathways. The IL-1 familymembers are primarily produced by innate immune cells and act on avariety of cell types during the immune response. Accordingly, in someembodiments the immunomodulatory domain is an IL-18 polypeptide.

Interleukin-2 (IL-2)

In some embodiments, the immunomodulatory fusion protein comprises amember of the IL-2 family operably linked to a collagen binding domain,optionally via a linker. In some embodiments, the member of the IL-2family is IL-2. Interleukin-2 (IL-2) is a cytokine that inducesproliferation of antigen-activated T cells and stimulates natural killer(NK) cells. The biological activity of IL-2 is mediated through amulti-subunit IL-2 receptor complex (IL-2R) of three polypeptidesubunits that span the cell membrane: p55 (IL-2Rα, the alpha subunit,also known as CD25 in humans), p75 (IL-2RP, the beta subunit, also knownas CD 122 in humans) and p64 (IL-2Rγ, the gamma subunit, also known asCD 132 in humans). T cell response to IL-2 depends on a variety offactors, including: (1) the concentration of IL-2; (2) the number ofIL-2R molecules on the cell surface; and (3) the number of IL-2Roccupied by IL-2 (i.e., the affinity of the binding interaction betweenIL-2 and IL-2R (Smith, “Cell Growth Signal Transduction is Quantal” InReceptor Activation by Antigens, Cytokines, Hormones, and Growth Factors766:263-271, 1995)). The IL-2:IL-2R complex is internalized upon ligandbinding and the different components undergo differential sorting.IL-2Rα is recycled to the cell surface, while IL-2 associated with theIL-2:IL-2RPγ complex is routed to the lysosome and degraded. Whenadministered as an intravenous (i.v.) bolus, IL-2 has a rapid systemicclearance (an initial clearance phase with a half-life of 12.9 minutesfollowed by a slower clearance phase with a half-life of 85 minutes)(Konrad et al., Cancer Res. 50:2009-2017, 1990).

Outcomes of systemic IL-2 administration in cancer patients are far fromideal. While 15 to 20 percent of patients respond objectively tohigh-dose IL-2, the great majority do not, and many suffer severe,life-threatening side effects, including nausea, confusion, hypotension,and septic shock. The severe toxicity associated with high-dose IL-2treatment is largely attributable to the activity of natural killer (NK)cells. NK cells express the intermediate-affinity receptor,IL-2RPyγ_(c), and thus are stimulated at nanomolar concentrations ofIL-2, which do in fact result in patient sera during high-dose IL-2therapy. Attempts to reduce serum concentration, and hence selectivelystimulate IL-2RaPγ_(c)-bearing cells, by reducing dose and adjustingdosing regimen have been attempted, and while less toxic, suchtreatments were also less efficacious. Given the toxicity issuesassociated with high dose IL-2 cancer therapy, numerous groups haveattempted to improve anti-cancer efficacy of IL-2 by simultaneouslyadministering therapeutic antibodies. Yet, such efforts have beenlargely unsuccessful, yielding no additional or limited clinical benefitcompared to IL-2 therapy alone. Accordingly, novel IL-2 therapies areneeded to more effectively combat various cancers.

In some embodiments, the linking of IL-2 to a collagen-binding domainlocalizes the cytokine to a cell, and therefore prevents systemictoxicity. Further, in some embodiments, when administered directly to atumor or lesion, the collagen-binding domain localizes the cytokine tothe tumor or lesion microenvironment, thereby preventing systemictoxicity associated with IL-2 treatment.

In some embodiments, the IL-2 is a human recombinant IL-2 such asProleukin® (aldesleukin). Proleukin® is a human recombinantinterleukin-2 product produced in E. coli. Proleukin® differs from thenative interleukin-2 in the following ways: a) it is not glycosylated;b) it has no N-terminal alanine; and c) it has serine substituted forcysteine at amino acid positions 125. Proleukin® exists as biologicallyactive, non-covalently bound microaggregates with an average size of 27recombinant interleukin-2 molecules. Proleukin® (aldesleukin) isadministered by intravenous infusion. In some embodiments, IL-2 iswild-type IL-2 (e.g., human IL-2 in its precursor form or mature IL-2.In some embodiments, IL-2 comprises the amino acid sequence set forth inSEQ ID NO: 1.

In certain embodiments, IL-2 is mutated such that it has an alteredaffinity (e.g., a higher affinity) for the IL-2R alpha receptor comparedwith unmodified IL-2. Site-directed mutagenesis can be used to isolateIL-2 mutants that exhibit high affinity binding to CD25, i.e., IL-2Rα,as compared to wild-type IL-2. Increasing the affinity of IL-2 forIL-2Rα at the cell surface will increase receptor occupancy within alimited range of IL-2 concentration, as well as raise the localconcentration of IL-2 at the cell surface.

In some embodiments, the disclosure features IL-2 mutants, which may be,but are not necessarily, substantially purified and which can functionas high affinity CD25 binders. IL-2 is a T cell growth factor thatinduces proliferation of antigen-activated T cells and stimulation of NKcells. Exemplary IL-2 mutants which are high affinity binders includethose described in WO2013/177187A2 (herein incorporated by reference inits entirety). Further exemplary IL-2 mutants with increased affinityfor CD25 are disclosed in U.S. Pat. No. 7,569,215, the contents of whichare incorporated herein by reference.

In some embodiments, the disclosure features IL-2 mutants with reducedbinding affinity to CD25 relative to wild-type IL-2. In someembodiments, the IL-2 mutant does not bind to CD25.

In some embodiments, IL-2 mutants comprise an amino acid sequence thatis at least 80% identical to SEQ ID NO: 1 that bind CD25. For example,some embodiments an IL-2 mutant has at least one mutation (e.g., adeletion, addition, or substitution of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more amino acid residues)that increases the affinity for the alpha subunit of the IL-2 receptorrelative to wild-type IL-2. It should be understood that mutationsidentified in mouse IL-2 may be made at corresponding residues in fulllength human IL-2 (nucleic acid sequence (accession: NM000586); aminoacid sequence (accession: P60568)) or human IL-2 without the signalpeptide. Accordingly, in some embodiments, the IL-2 is human IL-2. Inother embodiments, the IL-2 is a mutant human IL-2.

In some embodiments, IL-2 mutants are at least or about 50%, at least orabout 65%, at least or about 70%, at least or about 80%, at least orabout 85%, at least or about 87%, at least or about 90%, at least orabout 95%, at least or about 97%, at least or about 98%, or at least orabout 99% identical in amino acid sequence to wild-type IL-2 (in itsprecursor form or, preferably, the mature form). The mutation canconsist of a change in the number or content of amino acid residues. Forexample, the IL-2 mutants can have a greater or a lesser number of aminoacid residues than wild-type IL-2. Alternatively, or in addition, IL-2mutants can contain a substitution of one or more amino acid residuesthat are present in the wild-type IL-2.

By way of illustration, a polypeptide that includes an amino acidsequence that is at least 95% identical to a reference amino acidsequence of SEQ ID NO: 1 is a polypeptide that includes a sequence thatis identical to the reference sequence except for the inclusion of up tofive alterations of the reference amino acid of SEQ ID NO: 1. Forexample, up to 5% of the amino acid residues in the reference sequencemay be deleted or substituted with another amino acid, or a number ofamino acids up to 5% of the total amino acid residues in the referencesequence may be inserted into the reference sequence. These alterationsof the reference sequence may occur at the amino (N-) or carboxy (C-)terminal positions of the reference amino acid sequence or anywherebetween those terminal positions, interspersed either individually amongresidues in the reference sequence or in one or more contiguous groupswithin the reference sequence.

The substituted amino acid residue(s) can be, but are not necessarily,conservative substitutions, which typically include substitutions withinthe following groups: glycine, alanine; valine, isoleucine, leucine;aspartic acid, glutamic acid; asparagine, glutamine; serine, threonine;lysine, arginine; and phenylalanine, tyrosine. These mutations can be atamino acid residues that contact IL-2Rα.

Interleukin-12 (IL-12)

In some embodiments, the immunomodulatory fusion protein comprises anIL-12 polypeptide operably linked to a collagen binding domain,optionally via a linker. Interleukin-12 (IL-12) is a pro-inflammatorycytokine that plays an important role in innate and adaptive immunity.Gately, M K et al., Annu Rev Immunol. 16: 495-521 (1998). IL-12functions primarily as a 70 kDa heterodimeric protein consisting of twodisulfide-linked p35 and p40 subunits. The precursor form of the IL-12p40 subunit (NM_002187; P29460; also referred to as IL-12B, naturalkiller cell stimulatory factor 2, cytotoxic lymphocyte maturation factor2) is 328 amino acids in length, while its mature form is 306 aminoacids long. The precursor form of the IL-12 p35 subunit (NM_000882;P29459; also referred to as IL-12A, natural killer cell stimulatoryfactor 1, cytotoxic lymphocyte maturation factor 1) is 219 amino acidsin length and the mature form is 197 amino acids long. Id. The genes forthe IL-12 p35 and p40 subunits reside on different chromosomes and areregulated independently of each other. Gately, M K et al., Annu RevImmunol. 16: 495-521 (1998). Many different immune cells (e.g.,dendritic cells, macrophages, monocytes, neutrophils, and B cells)produce IL-12 upon antigenic stimuli. The active IL-12 heterodimer isformed following protein synthesis. Id.

Due to its ability to activate both NK cells and cytotoxic T cells,IL-12 protein has been studied as a promising anti-cancer therapeuticsince 1994. See Nastala, C. L. et al., J Immunol 153: 1697-1706 (1994).But despite high expectations, early clinical studies did not yieldsatisfactory results. Lasek W. et al., Cancer Immunol Immunother 63:419-435, 424 (2014). Repeated administration of IL-12, in most patients,led to adaptive response and a progressive decline of IL-12-inducedinterferon gamma (IFNγ) levels in blood. Id. Moreover, while it wasrecognized that IL-12-induced anti-cancer activity is largely mediatedby the secondary secretion of IFNγ, the concomitant induction of IFNγalong with other cytokines (e.g., TNF-α) or chemokines (IP-10 or MIG) byIL-12 caused severe toxicity. Id.

In addition to the negative feedback and toxicity, the marginal efficacyof the IL-12 therapy in clinical settings may be caused by the strongimmunosuppressive environment in humans. Id. To minimize IFNγ toxicityand improve IL-12 efficacy, scientists tried different approaches, suchas different dose and time protocols for IL-12 therapy. See Sacco, S. etal., Blood 90: 4473-4479 (1997); Leonard, J. P. et al., Blood 90:2541-2548 (1997); Coughlin, C. M. et al., Cancer Res. 57: 2460-2467(1997); Asselin-Paturel, C. et al., Cancer 91: 113-122 (2001); andSaudemont, A. et al., Leukemia 16: 1637-1644 (2002). Nonetheless, theseapproaches have not significantly impacted patient survival. Kang, W.K., et al., Human Gene Therapy 12: 671-684 (2001).

Membrane-anchored versions of IL-12 have been studied as a means ofreducing toxicity associated with systemic administration, usingretroviral and adenoviral vectors for expression in tumor cells. SeePan, W-Y. et al., Mol. Ther. 20(5): 927-937 (2012). But, the use ofviral vectors presents a potential health risk, since the underlyingviruses can act as oncogenes and the viral vectors can be immunogenic.

Accordingly, in some embodiments, the immunomodulatory fusion proteinsdisclosed herein comprise an IL-12 polypeptide operably linked to acollagen-binding domain. In some embodiments, the linking of an IL-12polypeptide to a collagen-binding domain localizes the cytokine to acell, and therefore prevents systemic toxicity. Further, in someembodiments, when administered directly to a tumor or lesion, thecollagen-binding domain localizes the cytokine to the tumor or lesionmicroenvironment, thereby preventing systemic toxicity.

In some embodiments, the IL-12 polypeptide comprises IL-12A (e.g., SEQID NO: 3). In some embodiments, the IL-12 polypeptide comprises IL-12B(e.g., SEQ ID NO: 2). In some embodiments, the IL-12 polypeptidecomprises both IL-12A and IL-12B.

In some embodiments, IL-12B is located N-terminal to IL-12A in the IL-12polypeptide. In some embodiments, IL-12A is located N-terminal to IL-12Bin the IL-12 polypeptide. The phrase “located N-terminal to” indicateslocation in a polypeptide with respect to other sequences in thepolypeptide in relation to the N-terminus of the polypeptide. Forexample, IL-12B that is “N-terminal to” IL-12A means that IL-12B islocated closer to the N-terminus of the IL-12 polypeptide than IL-12A.

In some embodiments, the IL-12 polypeptide comprises a singlepolypeptide chain comprising IL-12B and IL-12A, which are fused directlyto one another or are linked to one another by a linker (referred toherein as an “subunit linker”). Non-limiting examples of linkers aredisclosed elsewhere herein.

In some embodiments, the IL-12 polypeptide of the disclosure comprisesIL-12A and/or IL-12B that is a variant, that is a functional fragment,or that contains a substitution, an insertion and/or an addition, adeletion, and/or a covalent modification with respect to a wild-typeIL-12A or IL-12B sequence. In some embodiments, amino acid residueslocated at the carboxy, amino terminal, or internal regions of the IL-12polypeptide are deleted, thereby providing for fragments.

In some embodiments, the IL-12 polypeptide comprises a substitutionalvariant of an IL-12A and/or IL-12B amino acid sequence, which cancomprise one, two, three or more than three substitutions. In someembodiments, the substitutional variant can comprise one or moreconservative amino acids substitutions. In other embodiments, thevariant is an insertional variant. In other embodiments, the variant isa deletional variant.

As recognized by those skilled in the art, IL-12 protein fragments,functional protein domains, variants, and homologous proteins(orthologs) are also considered to be within the scope of the IL-12polypeptides of the disclosure. Nonlimiting examples of IL-12polypeptides suitable for use in the immunomodulatory fusion proteinsdisclosed herein are set forth in SEQ ID NOs: 2-3.

In some embodiments, the immunomodulatory fusion protein comprises anIL-12 polypeptide comprising the amino acid sequence set forth in SEQ IDNO: 2. In some embodiments, the immunomodulatory fusion proteincomprises an IL-12 polypeptide comprising the amino acid sequence setforth in SEQ ID NO: 3. In some embodiments, the immunomodulatory fusionprotein comprises an IL-12 polypeptide comprising the amino acidsequences set forth in SEQ ID NOs: 2 and 3.

Interleukin-15 (IL-15)

In some embodiments, the immunomodulatory fusion protein comprises anIL-15 polypeptide operably linked to a collagen binding domain,optionally via a linker. IL-15 is a member of the 4-helix bundle familyof cytokines and plays an important role in the development of aneffective immune response. Waldmann, T. A., Cancer Immunol. Res. 3:219-227 (2015). IL-15 is essential for the proper development of NKcells and long-term maintenance of memory CD8+ T cells. The IL-15 geneencodes a 162 amino acid preprotein having a signal peptide of 48 aminoacids, with the mature protein being 114 amino acids in length. Bamford,R. N., et al., Proc. Natl. Acad. Sci. USA 93: 2897-2902 (1996). Seealso, e.g., GenBank Accession Numbers NM_000585 for the Homo sapiensIL15 transcript variant 3 mRNA sequence and NP_000576 for thecorresponding IL15 isoform 1 preproprotein.

IL-15 shares certain structural similarity to interleukin-2 (IL-2). LikeIL-2, IL-15 signals through the IL-2 receptor beta chain (CD122) and thecommon gamma chain (CD132). But, unlike IL-2, IL-15 cannot effectivelybind CD122 and CD132 on its own. IL-15 must first bind to the IL-15alpha receptor subunit (IL-15Rα). The IL-15Rα gene encodes a 267 aminoacid preprotein having a signal peptide of 30 amino acids, with themature protein being 237 amino acids in length. See, e.g., GenBankAccession Numbers NM_002189 for the Homo sapiens IL-15Rα transcriptvariant 1 mRNA and NP_002180 for the Homo sapiens IL-15Rα isoform 1precursor amino acid sequence.

Human IL-15Rα is predominantly a transmembrane protein that binds toIL-15 on the surface of cells such as activated dendritic cells andmonocytes. Waldmann, T. A., Cancer Immunol. Res. 3: 219-227 (2015). Themembrane bound complex of IL-15/IL-15Rα then presents IL-15 in trans toCD122 and CD132 subunits. Accordingly, IL-15Rα is an essential componentof IL-15 activity.

To overcome the short half-life of systemically injected IL-15,pre-complexation of IL-15 with soluble recombinant IL-15Rα, resulting inIL-15 superagonist (IL-15SA) has been shown to enhance the systemicpotency of IL-15 by ˜50 fold, and also raises the half-life of thecytokine in serum following systemic injection to ˜20 hrs. (Stoklasek etal., J Immunol 177(9): 6072, 2006; Dubois et al., J Immunol 180(4):2099, 2008; Rubinstein et. al. Proc Natl Acad Sci USA 103(24): 9166,2006.)

Accordingly, in some embodiments, the immunomodulatory domain of theimmunomodulatory fusion protein is an IL-15 polypeptide. In someembodiments, the IL-15 polypeptide comprises the amino acid sequence setforth in SEQ ID NO: 5. In some embodiments, the IL-15 polypeptidecomprises the amino acid sequence set forth in SEQ ID NO: 4. In someembodiments, the IL-15 polypeptide is an IL-15 superagonist, comprisingIL-15 and IL-15Rα. In some embodiments, the IL-15 superagonist comprisesthe amino acid sequences set forth in SEQ ID NOs: 4 and 5.

B. Interferons

In some embodiments, the immunomodulatory domain suitable for use in theimmunomodulatory fusion proteins of the present disclosure, is aninterferon (IFN). Interferons comprise a family of secretory proteinsinduced in response to specific extracellular stimuli throughstimulation of toll-like receptors (TLRs). In some embodiments,interferons heighten anti-viral defenses of the immune system (e.g.,antigen presentation). Through high-affinity cell surface receptors,IFNs stimulate genes using signaling molecules. Interferons suitable foruse as an immunomodulatory domain of the immunomodulatory fusionproteins include, but are not limited to: IFN-gamma and IFN-alpha.

In some embodiments, the immunomodulatory fusion protein comprises anIFN-gamma polypeptide operably linked to a collagen-binding domain.IFN-gamma is produced by a variety of immune cells, such as activated Tcells and NK cells. IFN-gamma interacts with a specific receptor at thecell surface and activates signal transduction pathways that produceimmunomodulatory effects. Accordingly, in some embodiments, theimmunomodulatory domain is an IFN-gamma polypeptide. In someembodiments, the IFN-gamma polypeptide comprises the amino acid sequenceset forth in SEQ ID NO: 7.

In some embodiments, the immunomodulatory fusion protein comprises anIFN-alpha polypeptide operably linked to a collagen-binding domain.IFN-alpha is produced by B lymphocytes, null lymphocytes andmacrophages, and activates NK cells, along with having antiviral andantitumor activities. Accordingly, in some embodiments, theimmunomodulatory domain is an IFN-alpha polypeptide. In someembodiments, the IFN-alpha polypeptide comprises the amino acid sequenceset forth in SEQ ID NO: 6.

C. Immune Cell Differentiation Stimulating Factors

In some embodiments, the immunomodulatory domain suitable for use in theimmunomodulatory fusion proteins of the present disclosure, is an immunecell differentiation stimulating factor. In some embodiments, immunecell differentiation stimulating factors activate intracellularsignaling pathways that drive hematopoietic progenitor celldifferentiation, development and proliferation into specific subtypes ofimmune cells. Immune cell differentiation stimulating factors suitablefor use in the immunomodulatory fusion proteins disclosed hereininclude, but are not limited to: GM-CSF (granulocyte-macrophagecolony-stimulating factor), G-CSF (granulocyte colony-stimulatingfactor) and FLT3L (FMS-like tyrosine kinase 3 ligand).

In some embodiments, the immunomodulatory domain is a GM-CSFpolypeptide. GM-CSF is a monomeric glycoprotein secreted by macrophages,T cells, mast cells, NK cells, endothelial cells and fibroblasts. Inaddition to having a function of growth stimulation and differentiationon hematopoietic precursor cells, GM-CSF has a variety of effects onimmune cells expressing the GM-CSF receptor. In some embodiments, theGM-CSF polypeptide comprises the amino acid sequence set forth in SEQ IDNO: 27.

In some embodiments, the immunomodulatory domain is a FLT3L polypeptide.FLT3 is a receptor tyrosine kinase (RTK) which is expressed by immaturehematopoietic precursor cells. FLT3L is a transmembrane protein orsoluble protein and is expressed by a large number of cells, includinghematopoietic cells and stroma cells in the bone marrow. In combinationwith other growth factors, FLT3L stimulates proliferation anddevelopment of various cells types, including myeloid and lymphoidprecursor cells, dendritic cells and NK cells. In some embodiments, theFLT3L polypeptide comprises the amino acid sequence set forth in SEQ IDNO: 28.

In some embodiments, the immunomodulatory domain is an G-CSFpolypeptide. In some embodiments, G-CSF regulates proliferation,differentiation and functional activation of neutrophilic granulocytes.In some embodiments, the G-CSF polypeptide comprises the amino acidsequence set forth in SEQ ID NO: 29.

D. Chemokines

In some embodiments, the immunomodulatory domain suitable for use in theimmunomodulatory fusion proteins of the present disclosure, is achemokine. In some embodiments, chemokines are proteins that inducedirected chemotaxis of a responsive cell (e.g., leukocytes). In general,chemokines are grouped into four subfamilies: CXC, CC, (X)C, and CX3C.In the CXC chemokines, one amino acid separates the first two cysteines(“the CXC motif”). ELR+CXC chemokines are ligands for CXCR1 and/or CXCR2chemokine receptors, which are G-protein coupled seven transmembranedomain-type receptors that specifically bind ELR+CXC chemokines. Theseven human ELR+CXC chemokines are human Gro-alpha (also known asCXCL1), human Gro-beta (also known as CXCL2), human Gro-gamma (alsoknown as CXCL3), human ENA-78 (also known as CXCL5), human GCP-2 (alsoknown as CXCL6), human NAP-2 (also known as CXCL7), and human IL-8 (alsoknown as CXCL8). All ELR+CXC chemokines bind the CXCR2 receptor;moreover, some ELR+CXC chemokines bind both CXCR1 and CXCR2 receptors(i.e., CXCL6 and CXCL8), all of which contributes to redundancy in theactivation pathways. The five murine ELR+CXC chemokines are keratinocytechemoattractant (KC) (also known as CXCL1), Macrophage InflammatoryProtein-2 (MIP-2) (also known as CXCL2), dendritic cell inflammatoryprotein-1 (DCIP-1) (also known as CXCL3), lipopolysaccharide-induced CXCchemokine (LIX) (also known as CXCL5), and neutrophil activatingpeptide-2 (NAP-2) (also known as CXCL7).

Chemokines suitable for use in the immunomodulatory fusion proteindisclosed herein include, but are not limited to: LIF, M-CSF, MIP-2,MIP-1beta, KP (CXLC1), MIG (CXCL9), IP-10 (CXCL10), MCP-1, eotaxin,RANTES, LIX and MIP-1alpha.

Amino acids encoding exemplary chemokines suitable for use as animmunomodulatory domain for the immunomodulatory fusion proteindisclosed herein, are set forth below:

Amino acid sequence Chemokine (SEQ ID NO) LIF 30 M-CSF 31 MIP-2 32MIP-1beta 33 KP (CXCL1) 34 MIG (CXCL9) 35 IP-10 (CXCL10) 36 MCP-1 37Eotaxin 38 RANTES 39 LIX 40 MIP-1alpha 41

E. Tumor Necrosis Factor (TNF) Superfamily

In some embodiments, the immunomodulatory domain suitable for use in theimmunomodulatory fusion proteins of the present disclosure, is anextracellular domain of a member of the tumor necrosis factor (TNF)superfamily. The tumor necrosis factor super family of ligands andreceptors are a series of structurally homologous cell surface proteinsthat signal via forming trimeric clusters of ligand-receptor complexes.Ligation of activating TNF superfamily receptors can lead to a widerange of pro-immune responses, including proliferation, enhancedeffector function, and production of chemokines and cytokines. Someligands, such as Fas, can lead to the induction of apoptosis and areexpressed on the surface of immune cells. Additionally, other ligandsfunction as inhibitory receptors which weaken the immune response. Insome embodiments, the extracellular domain is derived from: TNF-alpha,LIGHT, LT-alpha, LT-beta, BTLA, CD160, CD40L, FasL, CD30L, 4-1BBL,CD27L, OX40L, TWEAK, APRIL, BAFF, RANKL, TRAIL, EDA1, EDA2 or GITRL. Theextracellular domain is capable of binding the selected TNF superfamilymember's receptor, thereby inducing or stimulating an immune response.

The following table shows the receptor corresponding to the derivedextracellular domain:

Amino acid sequence of ligand extracellular Ligand Receptor domain (SEQID NO) TNF-alpha TNFR1, TNFR2 51 LIGHT HEVM, LT-betaR 52 LT-alpha TNFR1,TNFR2, HEVM 53 LT-beta LT-BetaR 54 CD160 HVEM 56 CD40L CD40 57 FasL Fas58 CD30L CD30 59 4-1BBL 4-1BB 60 CD27L CD27 61 OX40L OX40 62 TWEAK Fn1463 APRIL BCMA, TACI 64 BAFF BCMA, TACI, BAFFR 65 RANKL RANK, OPG 66TRAIL OPG, TRAIL R1 (DR4), TRAIL 67 R2 (DR5), DcR1, DcR2 EDA1 EDAR 68EDA2 XEDAR 69 GITRL GITR 70

F. CD28 Family

In some embodiments, the immunomodulatory domain suitable for use in theimmunomodulatory fusion proteins of the present disclosure, is anextracellular domain of a member of the CD28 family. The CD28 family isa family of inhibitory (PD 1, CTLA-4) and activating (CD28, ICOS)receptors that bind to members of the B7 family of ligands. CD28 is aco-stimulatory receptor that provides the second signal required toactivate naive T cells (along with ligation of the TCR) and has twonatural ligands, CD80 and CD86. CD28 signaling can serve to increaseproliferation, effector function, and anti-apoptotic signaling. CD28signaling has recently been shown to be required in effective PD1/PDL1blockade. ICOS (Inducible T cell Costimulator) is a closely relatedsurface receptor that is expressed on activated T cells and displayssimilar functions as CD28.

Accordingly, in some embodiments, the immunomodulatory domain is anextracellular domain of CD80 (B7-1). In some embodiments, theimmunomodulatory domain comprises the amino acid sequence set forth inSEQ ID NO: 71.

Accordingly, in some embodiments, the immunomodulatory domain is anextracellular domain of CD86 (B7-2), capable of binding CD28. In someembodiments, the immunomodulatory domain comprises the amino acidsequence set forth in SEQ ID NO: 72.

Accordingly, in some embodiments, the immunomodulatory domain is anextracellular domain of ICOSLG. In some embodiments, theimmunomodulatory domain comprises the amino acid sequence set forth inSEQ ID NO: 73.

G. Agonistic Antibodies

In some embodiments, the immunomodulatory domain suitable for use in theimmunomodulatory fusion proteins of the present disclosure, is anagonistic antibody, or antigen binding fragment thereof. Agonisticantibodies activate their target of interest, in contrast toantagonistic antibodies which block the function of their target. Insome embodiments, the agonistic antibodies, or antigen binding fragmentsthereof, bind to immune activating receptors.

In some embodiments, immune activating receptors include, but are notlimited to: tumor necrosis factor (TNF) receptors, CD28 family members,T-cell receptors (TCRs), Killer cell Ig-Like receptors (KIRs), LeukocyteIg-Like receptors (LIRs), CD94/NKG2 receptors, Fc receptors, signalinglymphocytic activation molecules (SLAMs), and activating Siglecreceptors.

Tumor Necrosis Factor (TNF) Superfamily

In some embodiments, the immunomodulatory domain suitable for use in theimmunomodulatory fusion proteins of the present disclosure, is anagonistic antibody, or antigen binding fragment thereof, that binds to atumor necrosis factor (TNF) superfamily member receptor. The TNFsuperfamily is described supra. For example, in some embodiments, theimmunomodulatory domain is an agonistic antibody, or antigen bindingfragment, that binds to TNFR1, thereby activating the receptor.

The following table provides a list of TNF superfamily member receptorsthat agonistic antibodies, or antigen binding fragments thereof, can begenerated to target, suitable for use in the immunomodulatory fusionprotein described herein:

Ligand Receptor Receptor Uniprot KB TNF-alpha TNFR1 P19438 TNFR2 P20333LIGHT HEVM Q92956 LT-betaR Q06643 LT-alpha TNFR1 P19438 TNFR2 P20333HEVM Q92956 LT-beta LT-BetaR Q06643 CD160 HVEM Q92956 CD40L CD40 P25942FasL Fas P25445 CD30L CD30 P28908 4-1BBL 4-1BB Q07011 CD27L CD27 P26842OX40L OX40 P43489 TWEAK Fn14 Q9NP84 APRIL BCMA Q02223 TACI O14836 BAFFBCMA Q02223 TACI O14836 BAFFR Q96RJ3 RANKL RANK Q9Y6Q6 OPG O00300 TRAILOPG O00300 TRAIL R1 (DR4) O00220 TRAIL R2 (DR5) O14763 DcR1 O14798 DcR2Q9UBN6 EDA1 EDAR Q9UNE0 EDA2 XEDAR Q9HAV5 GITRL GITR Q9Y5U5

In some embodiments, the immunomodulatory domain is an anti-4-1BBagonist antibody. In some embodiments, the immunomodulatory domain is ananti-OX40 agonist antibody. In some embodiments, the immunomodulatorydomain is a CD40 agonist antibody.

CD28 Receptor Superfamily

In some embodiments, the immunomodulatory domain suitable for use in theimmunomodulatory fusion proteins of the present disclosure, is anagonistic antibody, or antigen binding fragment thereof, that binds to aCD28 superfamily receptor. The CD28 superfamily is described supra. Forexample, in some embodiments, the immunomodulatory domain is anagonistic antibody, or antigen binding fragment, that binds to CD28,thereby activating the receptor.

The following table provides a list of CD28 superfamily member receptorsthat agonistic antibodies, or antigen binding fragments thereof, can begenerated to target, suitable for use in the immunomodulatory fusionprotein described herein:

Ligand Receptor Receptor Uniprot KB CD80 (B7-1) CD28 P10747 CD86 (B7-2)CD28 P10747 ICOSLG ICOS Q9Y6W8

T Cell Receptor (TCR) Complex

In some embodiments, the immunomodulatory domain suitable for use in theimmunomodulatory fusion proteins of the present disclosure, is anagonistic antibody, or antigen binding fragment thereof, that binds to aT-cell Receptor (TCR) complex. The T-cell Receptor (TCR) is the cellsurface receptor responsible for imparting antigen specificity toT-cells. Each TCR is specific for a particular peptide presented eitherby MHC Class I (for CD8+ T cells) or MHC Class II (for CD4+ T cells).For naive T cells, ligation of the TCR provides the first of two signalsrequired to activate the T cell. TCR ligation of CD8+ T cells leads todeath of the cell displaying the cognate pMHC (and potentially bystandercells) via release of soluble factors, such as perforin and granzyme B,as well as upregulation of apoptosis inducing ligands, such as Fasligand. For CD4+ helper T cells, ligation of the TCR with its cognatepMHC results in the release of cytokines,

Accordingly, in some embodiments, the immunomodulatory domain is anagonistic antibody, or antigen binding fragment thereof, that binds to aTCR. For example, in some embodiments, the immunomodulatory domain is anagonistic antibody, or antigen binding fragment, that binds to CD3γ,thereby activating the receptor.

The following table provides a list of members of TCR complexes thatagonistic antibodies, or antigen binding fragments thereof, can begenerated to target, suitable for use in the immunomodulatory fusionprotein described herein:

TCR Binder TCR Complex Member Member Uniprot KB pMHC CD3γ P09693 pMHCCD3δ P04234 pMHC CD3ζ P20963 pMHC CD3ε P07766

Killer Cell Ig-Like Receptor (KIR)

In some embodiments, the immunomodulatory domain suitable for use in theimmunomodulatory fusion proteins of the present disclosure, is anagonistic antibody, or antigen binding fragment thereof, that binds to aKiller Cell Ig-Like Receptor (KIR). The killer cell immunoglobulin likereceptor (KIR) is a family of receptors expressed mainly on NK cells andon some subsets of T cells. These receptors are primarily responsiblethrough recognition of self (and therefore inhibitory function), bybinding to MHC class I (HLA-A, HLA-B, and HLA-C) molecules. Thesereceptors can be either activating or inhibitory, depending on thelength of the cytoplasmic tail. Inhibitory receptors have a longer tailand contain an ITIM domain. Activating KIRs have a shorter cytoplasmicdomain and associate with DAP12 to mediate signaling.

Activating KIRs are provided in the table below, in which agonisticantibodies, or antigen binding fragments thereof, can be generated totarget, suitable for use in the immunomodulatory fusion proteindescribed herein:

Ligand Receptor Receptor Uniprot KB HLA molecules KIR 2DS1 Q14954 HLAmolecules KIR 2DS2 P43631 HLA molecules KIR 2DS3 Q14952 HLA moleculesKIR 2DS4 P43632 HLA molecules KIR 2DS5 Q14953 HLA molecules KIR 3DS1Q14943

Leukocyte Ig-Like Receptor (LIR)

In some embodiments, the immunomodulatory domain suitable for use in theimmunomodulatory fusion proteins of the present disclosure, is anagonistic antibody, or antigen binding fragment thereof, that binds to aleukocyte Ig-Like receptor (LIR). LIR receptors are a class of immunereceptors expressed primarily on innate immune cells. Their primaryligand is MHC Class I molecules and they largely exhibit inhibitoryfunctions, although some have activating functions. LIRA2, for example,acts as an innate sensor of immunoglobulin fragments that have beencleaved by microbial proteases.

In some embodiments, the immunomodulatory domain is an agonisticantibody, or antigen binding fragment thereof, that binds to LIRA2. Insome embodiments, antibodies capable of binding to LIRA2 can begenerated based on Uniprot ID Q8N149.

CD94/NKG2 Receptor Family

In some embodiments, the immunomodulatory domain suitable for use in theimmunomodulatory fusion proteins of the present disclosure, is anagonistic antibody, or antigen binding fragment thereof, that binds to aCD94/NKG2 receptor. CD94/NKG2 are heterodimer C-type lectin receptorsthat are expressed on the surface of NK cells and some subsets of CD8 Tcells. They bind to HLA-E molecules (non-classical MHC Class Imolecules) and can transmit both inhibitory and activating signals to NKCells. Inhibitory receptors contain ITIM domains in their cytoplasmictails, while activating receptors associate with DAP12 and DAP10 whichcontain ITAM domains.

Activating CD94/NKG2 receptors are provided in the table below, in whichagonistic antibodies, or antigen binding fragments thereof, can begenerated to target, suitable for use in the immunomodulatory fusionprotein described herein.

In some embodiments, the immunomodulatory domain suitable for use in theimmunomodulatory fusion proteins of the present disclosure is anextracellular domain of a CD94/NKG2 ligand. The following table showsthe receptor corresponding to the derived extracellular domain.

Amino acid sequence Receptor of ligand extracellular Ligand ReceptorUniprot KB domain (SEQ ID NO) MICA CD94 Q13241 74 NKG2D P26718 MICB CD94Q13241 75 NKG2D P26718 ULBP1 CD94 Q13241 76 NKG2D P26718 ULBP2 CD94Q13241 77 NKG2D P26718 ULBP3 CD94 Q13241 78 NKG2D P26718 ULBP4 CD94Q13241 79 NKG2D P26718 ULBP5, isoform 1 CD94 Q13241 80 NKG2D P26718ULBP5, isoform 2 CD94 Q13241 81 NKG2D P26718 ULBP6 NKG2D P26718 82 NKG2CP26717 NKG2E Q07444 NKG2H O43908 CD94 Q13241

Fc Receptors

In some embodiments, the immunomodulatory domain suitable for use in theimmunomodulatory fusion proteins of the present disclosure, is anagonistic antibody, or antigen binding fragment thereof, that binds toan Fc receptor. Fc receptors are immune cell receptors expressedprimarily on innate immune cells which bind to the constant region ofantibodies and elicit a wide range of functions. Fc receptors are almostexclusively activating (except for Fc γ RIIB, which transmits inhibitorysignals). Fc receptor ligation can lead to ADCC, phagocytosis,degranulation, and the transmission of activating signals which increaseeffector function.

The following table provides a list of Fc receptors that agonisticantibodies, or antigen binding fragments thereof, can be generated totarget, suitable for use in the immunomodulatory fusion proteindescribed herein:

Ligand Receptor Receptor Uniprot KB IgG FcγRI P12314 IgG FcγRIIC P31995IgG FcγRIIIA P12318 IgG FcγRIIIB P31994 IgE FcεRI P30273 IgE FcεRIIP06734 IgA FcαR P24071 IgA/IgM FcμR Q8WWV6

Signaling Lymphocytic Activation Molecules (SLAM)

In some embodiments, the immunomodulatory domain suitable for use in theimmunomodulatory fusion proteins of the present disclosure, is anagonistic antibody, or antigen binding fragment thereof, that binds to asignaling lymphocytic activation molecule (SLAM) receptor. SLAMreceptors are a series of molecules that function both as receptors andligands. SLAM molecules interact with one another on adjacent cells tosend either activating or inhibitory signals. SLAM molecules containImmunoreceptor Tyrosine based Swith motifs in their cytoplasmic tails,allowing them to associate with both activating and inhibitory signalingmolecules intracellularly.

The following table provides a list of SLAM receptors that agonisticantibodies, or antigen binding fragments thereof, can be generated totarget, suitable for use in the immunomodulatory fusion proteindescribed herein.

In some embodiments, the immunomodulatory domain suitable for use in theimmunomodulatory fusion proteins of the present disclosure is anextracellular domain of a SLAM ligand. The following table shows thereceptor corresponding to the derived extracellular domain.

Amino acid sequence of ligand extracellular Ligand Receptor ReceptorUniprot KB domain (SEQ ID NO) SLAMF1 SLAMF1 Q13291 83 SLAMF2 SLAMF2P09326 84 SLAMF3 SLAMF3 Q9HBG7 85 SLAMF4 SLAMF4 Q9BZW8 86 SLAMF5 SLAMF5Q9UIB8 87 SLAMF6 SLAMF6 Q96DU3 88 SLAMF7 SLAMF7 Q9NQ25 89

Siglec Family Receptors

In some embodiments, the immunomodulatory domain suitable for use in theimmunomodulatory fusion proteins of the present disclosure, is anagonistic antibody, or antigen binding fragment thereof, that binds to aSiglec family receptor. Siglecs are a family of surface receptors foundmainly on immune cells that are part of the lectin family (sugar bindingproteins). These receptors bind to sialic acid containing ligands. Thesereceptors function mainly as inhibitory receptors on a wide range ofimmune cell types, although some (siglec 14, 15, and 16) contain an ITAMactivating domain.

Activating Siglec receptors are provided in the table below, in whichagonistic antibodies, or antigen binding fragments thereof, can begenerated to target, suitable for use in the immunomodulatory fusionprotein described herein:

Receptor Receptor Uniprot KB Siglec 14 Q08ET2 Siglec 15 Q6ZMC9 Siglec 16A6NMB1

H. Antagonistic Antibodies

In some embodiments, the immunomodulatory domain suitable for use in theimmunomodulatory fusion proteins of the present disclosure, is anantagonistic antibody, or antigen binding fragment thereof. Antagonisticantibodies block the function of their target. In some embodiments, theantagonistic antibodies, or antigen binding fragments thereof, bind toimmune inhibitory receptors, thereby allowing for the induction of animmune response. In some embodiments, the antagonistic antibodies, orantigen binding fragments thereof, bind to immune inhibitory ligands,thereby allowing for the induction of an immune response. In someembodiments, immune inhibitor receptors and ligands include, but are notlimited to: CD28 receptors, tumor necrosis factor (TNF) superfamilyreceptors, Siglec receptors, CD94/NKG2 receptors, Leukocyte Ig-Likereceptors (LIRs), Killer Cell Ig-Like receptors (KIRs), Fc receptors,adenosine pathway molecules, other checkpoint inhibitors, and LAIR1.

CD28 Molecules

In some embodiments, the immunomodulatory domain suitable for use in theimmunomodulatory fusion proteins of the present disclosure, is anantagonistic antibody, or antigen binding fragment thereof, that binds aCD28 molecule. As described supra, the CD28 family includes bothactivating and inhibitory molecules. Accordingly, in some embodiments,antagonizing the inhibitory molecules results in an induction orstimulation of immune responses.

The following table provides a list of CD28 molecules that antagonisticantibodies, or antigen binding fragments thereof, can be generated totarget, suitable for use in the immunomodulatory fusion proteindescribed herein.

Molecule Molecule Uniprot KB PD1 Q15116 PDL1 Q9NZQ7 PDL2 Q9BQ51 CTLA-4P16410 B7-H4 Q7Z7D3 B7-H3 Q5ZPR3

In some embodiments, the immunomodulatory domain is an antagonisticantibody, or antigen binding fragment thereof, that binds PD-1. In someembodiments, the immunomodulatory domain is an antagonistic antibody, orantigen binding fragment thereof, that binds PD-L1. In some embodiments,the immunomodulatory domain is an antagonistic antibody, or antigenbinding fragment thereof, that binds CTLA-4.

TNF Superfamily Molecules

In some embodiments, the immunomodulatory domain suitable for use in theimmunomodulatory fusion proteins of the present disclosure, is anantagonistic antibody, or antigen binding fragment thereof, that binds aTNF superfamily member. As described supra, the TNF superfamily includesboth activating and inhibitory molecules. Accordingly, in someembodiments, antagonizing the inhibitory molecules results in aninduction or stimulation of immune responses.

The following table provides a list of TNF superfamily molecules thatantagonistic antibodies, or antigen binding fragments thereof, can begenerated to target, suitable for use in the immunomodulatory fusionprotein described herein.

Molecule Molecule Uniprot KB TIGIT Q495A1 BTLA Q7Z6A9

Siglec Receptors

In some embodiments, the immunomodulatory domain suitable for use in theimmunomodulatory fusion proteins of the present disclosure, is anantagonistic antibody, or antigen binding fragment thereof, that binds aSiglec receptor. As described supra, the Siglec family includes bothactivating and inhibitory molecules. Accordingly, in some embodiments,antagonizing the inhibitory molecules results in an induction orstimulation of immune responses.

The following table provides a list of Siglec receptors thatantagonistic antibodies, or antigen binding fragments thereof, can begenerated to target, suitable for use in the immunomodulatory fusionprotein described herein.

Receptor Receptor Uniprot KB Siglec 1 (siualoadhesion) Q9BZZ2 Siglec 2(CD22) P20273 Siglec 3 (CD33) P20138 Siglec 4a (MAG) P20916 Siglec 5O15389 Siglec 6 O43699 Siglec 7 Q9Y286 Siglec 8 Q9NYZ4 Siglec 9 Q9Y336Siglec 10 Q96LC7 Siglec 11 Q96RL6 Siglec 12 Q96PQ1

CD94/NKG2 Receptors

In some embodiments, the immunomodulatory domain suitable for use in theimmunomodulatory fusion proteins of the present disclosure, is anantagonistic antibody, or antigen binding fragment thereof, that binds aCD94/NKG2 receptors. As described supra, the CD94/NKG2 family includesboth activating and inhibitory molecules. Accordingly, in someembodiments, antagonizing the inhibitory molecules results in aninduction or stimulation of immune responses.

Accordingly, in some embodiments, the immunomodulatory domain is anantagonistic antibody, or antigen binding fragment thereof, that bindsCD94/NKG2A. In some embodiments, such antibodies are generated based onUniProt ID P26715.

In some embodiments, the immunomodulatory domain is an antagonisticantibody, or antigen binding fragment thereof, that binds CD94/NKG2B. Insome embodiments, such antibodies are generated based on UniProt IDQ13241.

Leukocyte Ig-Like Receptors (LIRs)

In some embodiments, the immunomodulatory domain suitable for use in theimmunomodulatory fusion proteins of the present disclosure, is anantagonistic antibody, or antigen binding fragment thereof, that binds aLeukocyte Ig-Like Receptors (LIR). As described supra, the LIR familyincludes both activating and inhibitory molecules. Accordingly, in someembodiments, antagonizing the inhibitory molecules results in aninduction or stimulation of immune responses.

The following table provides a list of LIRs that antagonisticantibodies, or antigen binding fragments thereof, can be generated totarget, suitable for use in the immunomodulatory fusion proteindescribed herein.

Receptor Receptor Uniprot KB LIRB1 Q8NHL6 LIRB2 Q8N423 LIRB3 O75022LIRB4 Q8NHJ6

Killer Cell Ig-Like Receptors (KIRs)

In some embodiments, the immunomodulatory domain suitable for use in theimmunomodulatory fusion proteins of the present disclosure, is anantagonistic antibody, or antigen binding fragment thereof, that binds aKiller Cell Ig-Like Receptor (KIR). As described supra, the KIR familyincludes both activating and inhibitory molecules. Accordingly, in someembodiments, antagonizing the inhibitory molecules results in aninduction or stimulation of immune responses.

The following table provides a list of KIRs that antagonisticantibodies, or antigen binding fragments thereof, can be generated totarget, suitable for use in the immunomodulatory fusion proteindescribed herein.

Receptor Receptor Uniprot KB KIR 2DL1 P43626 KIR 2DL2 P43627 KIR 2DL3P43628 KIR 2DL4 Q99706 KIR 2DL5A Q8N109 KIR 2DL5B Q8NHK3 KIR 3DL1 P43629KIR 3DL2 P43630 KIR 3DL3 Q8N743

Fc Receptors

In some embodiments, the immunomodulatory domain suitable for use in theimmunomodulatory fusion proteins of the present disclosure, is anantagonistic antibody, or antigen binding fragment thereof, that bindsan Fc receptor. As described supra, the family of Fc receptors includesboth activating and inhibitory molecules. Accordingly, in someembodiments, antagonizing the inhibitory molecules results in aninduction or stimulation of immune responses.

In some embodiments, the inhibitor Fc receptor is Fc γ RIIB. In someembodiments, the immunomodulatory domain is an antagonistic antibody, orantigen binding fragment thereof, that binds Fc γ RIIB. In someembodiments, such antibodies are generated based on UniProt ID P31994.

Adenosine Pathway Molecules

In some embodiments, the immunomodulatory domain suitable for use in theimmunomodulatory fusion proteins of the present disclosure, is anantagonistic antibody, or antigen binding fragment thereof, that binds amember of the adenosine pathway. For example, CD39 and CD73 are enzymesexpressed on the surface of cells which catalyze the transformation ofATP into adenosine. Extracellular ATP is a danger molecule which elicitsan immune response, while adenosine is immunosuppressive. Thesemolecules contribute to a locally immunosuppressive environment bygenerating adenosine.

Accordingly, in some embodiments, the immunomodulatory domain is anantagonistic antibody, or antigen binding fragment thereof, that bindsCD39. In some embodiments, such antibodies are generated based onUniProt ID P49961.

In some embodiments, the immunomodulatory domain is an antagonisticantibody, or antigen binding fragment thereof, that binds CD73. In someembodiments, such antibodies are generated based on UniProt ID P21589.

Other Checkpoint Inhibitors

In some embodiments, the immunomodulatory domain suitable for use in theimmunomodulatory fusion proteins of the present disclosure, is anantagonistic antibody, or antigen binding fragment thereof, that bindsan immune checkpoint inhibitor. In some embodiments, by antagonizingsuch immune checkpoint inhibitors, an immune response is induced orstimulated.

The following table provides a list of immune checkpoint inhibitors thatantagonistic antibodies, or antigen binding fragments thereof, can begenerated to target, suitable for use in the immunomodulatory fusionprotein described herein.

Molecule Molecule Uniprot KB VISTA Q9H7M9 TIM-3 Q8TDQ0 LAG-3 P18627 CD47Q08722 SIRPα P78324

III. Linkers

In some embodiments, the immunomodulatory fusion protein comprises animmunomodulatory domain operably linked to a collagen-binding proteinvia a linker. In some embodiments, the linker between theimmunomodulatory domain and the collagen-binding protein provides asteric separation such that the immunomodulatory domain retains itsactivity (e.g., promote receptor/ligand engagement). In someembodiments, the linker between the immunomodulatory domain and thecollagen-binding protein is of sufficient length or mass to reduceadsorption of the immunomodulatory domain onto collagen fibrils. Methodsfor measuring adsorption are known to those of skill in the art. Forexample, adsorption can be measured by ellipsometry (ELM), surfaceplasmon resonance (SPR), optical waveguide lightmode spectroscopy(OWLS), attenuated total internal reflectance-infrared spectroscopy(ATR-IR), circular dichroism spectroscopy (CD), total internalreflectance-infrared spectroscopy (TIRF), and other high resolutionmicroscopy techniques. In some embodiments, these methods show thespatial arrangement between the domains of the immunomodulatory fusionprotein.

In some embodiments, the linker between the immunomodulatory domain andthe collagen-binding protein provides sufficient molecular weight toslow or reduce diffusion from the tissue. Methods for measuringdiffusion from the tissue are known to those of skill in the art. Forexample, diffusion can be measured by in vivo imagining, or viamicroscopy of tissue sections over time. Exemplary methods are describedin at least Schmidt & Wittrup, Mol Canc Ther. 2009′ and Wittrup et al.,Methods in Enzymol 2012, each of which is herein incorporated byreference in their entirety.

In some embodiments, the linker is a hydrophilic polypeptide comprising“N” amino acids in length, wherein N=1-1000, 50-800, 100-600, or200-500.

A. Serum Albumin

In some embodiments, the linker is a serum albumin, or fragmentsthereof. Methods of fusing serum albumin to proteins are disclosed in,e.g., US2010/0144599, US2007/0048282, and US2011/0020345, which areherein incorporated by reference in their entirety. In some embodiments,the linker is human serum albumin (HSA), or variants or fragmentsthereof, such as those disclosed in U.S. Pat. No. 5,876,969, WO2011/124718, WO 2013/075066, and WO 2011/0514789.

Suitable albumins for use in the immunomodulatory fusion proteins can befrom human, primate, rodent, bovine, equine, donkey, rabbit, goat,sheep, dog, chicken, or pig. In some embodiments, the albumin is a serumalbumin, for example, a human serum albumin (SEQ ID NO: 42, primateserum albumin (e.g., chimpanzee serum albumin, gorilla serum albumin),rodent serum albumin (e.g., hamster serum albumin, guinea pig serumalbumin, mouse albumin and rat serum albumin), bovine serum albumin,equine serum albumin, donkey serum albumin, rabbit serum albumin, goatserum albumin, sheep serum albumin, dog serum albumin, chicken serumalbumin and pig serum albumin.

In some embodiments, the albumin, or a variant or fragment thereof, hasa sequence identity to the sequence of wild-type HSA as set forth in SEQID NO: 42 of at least 50%, such as at least 60%, at least 70%, at least80%, at least 85%, at least 86%, at least 87%, at least 88%, at least89%, at least 90%, at least 91%, at least 92%, at least 93%, at least94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least99%.

In some embodiments, the number of alterations, e.g., substitutions,insertions, or deletions, in an albumin variants is 1-20, e.g., 1-10 and1-5, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 alterations compared to thecorresponding wild-type albumin (e.g., HSA).

In some embodiments, fragments of albumin, or fragments of variantsthereof, are suitable for use in the immunomodulatory fusion proteins.Exemplary albumin fragments are disclosed in WO 2011/124718. In someembodiments, a fragment of albumin (e.g., a fragment of HSA) is at least20 amino acids in length, such as at least 40 amino acids, at least 60amino acids, at least 80 amino acids, at least 100 amino acids, at least150 amino acids, at least 200 amino acids, at least 300 amino acids, atleast 400 amino acids, or at least 500 amino acids in length.

In some embodiments, an albumin fragment may comprise at least one wholesub-domain of albumin. Domains of HSA have been expressed as recombinantproteins (Dockal et al., JBC 1999; 274:9303-10), where domain I wasdefined as consisting of amino acids 1-197 (SEQ ID NO: 116), domain IIwas defined as consisting of amino acids 189-385 (SEQ ID NO: 117), anddomain III was defined as consisting of amino acids 381-585 (SEQ ID NO:118) of HSA (SEQ ID NO: 42). Partial overlap of the domains occurs giventhe extended α-helix structure (h10-h1) which exists between domains Iand II, and between domains II and III (Peters, 1996, op. cit, Table2-4). HSA also comprises six sub-domains (sub-domains IA, IB, NA, NB,INA and NIB). Sub-domain IA comprises amino acids 6-105, sub-domain IBcomprises amino acids 120-177, sub-domain NA comprises amino acids200-291, sub-domain NB comprises amino acids 316-369, sub-domain INAcomprises amino acids 392-491 and sub-domain NIB comprises amino acids512-583 of SEQ ID NO: 42

In some embodiments, a fragment comprises a whole or part of one or moredomains or sub-domains as defined above, or any combination of thosedomains and/or sub-domains. In some embodiments, an albumin fragmentcomprises at least 50, 60, 70, 75, 80, 85, 90, 95, 96, 97, 98, or 99% ofan albumin or of a domain of an albumin, or a variant or fragmentthereof.

B. Fc Domains

In some embodiments, the linker suitable for use in the immunomodulatoryfusion protein described herein is an Fc domain. In some embodiments,the Fc domain is a component of the agonist or antagonist antibodiesdescribed supra, and therefore a separate Fc domain is not needed.

In certain embodiments, the Fc domain comprises the amino acid sequenceset forth in SEQ ID NO: 115. In some embodiments, the Fc domain does notcontain a variable region that binds to antigen. In some embodiments,the Fc domain contains a variable region that binds to antigen. Fcdomains suitable for the immunomodulatory fusion proteins disclosedherein may be obtained from a number of different sources. In certainembodiments, an Fc domain is derived from a human immunoglobulin. Incertain embodiments, the Fc domain is from a human IgG1 constant region.The Fc domain of human IgG1 is set forth in SEQ ID NO: 115. It isunderstood, however, that the Fc domain may be derived from animmunoglobulin of another mammalian species, including for example, arodent (e.g. a mouse, rat, rabbit, guinea pig) or non-human primate(e.g. chimpanzee, macaque) species. Moreover, the Fc domain or portionthereof may be derived from any immunoglobulin class, including IgM,IgG, IgD, IgA, and IgE, and any immunoglobulin isotype, including IgG1,IgG2, IgG3, and IgG4.

In some embodiments, the immunomodulatory fusion protein comprises amutant Fc domain. In some embodiments, the immunomodulatory fusionprotein comprises a mutant, IgG1 Fc domain. In some embodiments, amutant Fc domain comprises one or more mutations in the hinge, CH2,and/or CH3 domains. In some aspects, a mutant Fc domain includes a D265Amutation.

A variety of Fc domain gene sequences (e.g., mouse and human constantregion gene sequences) are available in the form of publicly accessibledeposits. Constant region domains comprising an Fc domain sequence canbe selected lacking a particular effector function and/or with aparticular modification to reduce immunogenicity. Many sequences ofantibodies and antibody-encoding genes have been published and suitableFc domain sequences (e.g. hinge, CH2, and/or CH3 sequences, or portionsthereof) can be derived from these sequences using art recognizedtechniques. The genetic material obtained using any of the foregoingmethods may then be altered or synthesized to obtain polypeptidessuitable for use in the methods disclosed herein. It will further beappreciated that the scope of this disclosure encompasses alleles,variants and mutations of constant region DNA sequences.

Fc domain sequences can be cloned, e.g., using the polymerase chainreaction and primers which are selected to amplify the domain ofinterest. To clone an Fc domain sequence from an antibody, mRNA can beisolated from hybridoma, spleen, or lymph cells, reverse transcribedinto DNA, and antibody genes amplified by PCR. PCR amplification methodsare described in detail in U.S. Pat. Nos. 4,683,195; 4,683,202;4,800,159; 4,965,188; and in, e.g., “PCR Protocols: A Guide to Methodsand Applications” Innis et al. eds., Academic Press, San Diego, Calif.(1990); Ho et al. 1989. Gene 77:51; Horton et al. 1993. Methods Enzymol.217:270. PCR may be initiated by consensus constant region primers or bymore specific primers based on the published heavy and light chain DNAand amino acid sequences. As discussed above, PCR also may be used toisolate DNA clones encoding the antibody light and heavy chains. In thiscase the libraries may be screened by consensus primers or largerhomologous probes, such as mouse constant region probes. Numerous primersets suitable for amplification of antibody genes are known in the art(e.g., 5′ primers based on the N-terminal sequence of purifiedantibodies (Benhar and Pastan. 1994. Protein Engineering 7: 1509); rapidamplification of cDNA ends (Ruberti, F. et al. 1994. J. Immunol. Methods173:33); antibody leader sequences (Larrick et al. Biochem Biophys ResCommun 1989; 160: 1250). The cloning of antibody sequences is furtherdescribed in Newman et al., U.S. Pat. No. 5,658,570, filed Jan. 25,1995, which is herein incorporated by reference.

In some embodiments, the immunomodulatory fusion protein disclosedcomprises one or more Fc domains (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, ormore Fc domains). In certain embodiments, the Fc domains may be ofdifferent types. In certain embodiments, at least one Fc domain presentin the immunomodulatory fusion protein comprises a hinge domain orportion thereof. In certain embodiments, the immunomodulatory fusionprotein comprises at least one Fc domain which comprises at least oneCH2 domain or portion thereof. In certain embodiments, theimmunomodulatory fusion protein comprises at least one Fc domain whichcomprises at least one CH3 domain or portion thereof. In certainembodiments, the immunomodulatory fusion protein comprises at least oneFc domain which comprises at least one CH4 domain or portion thereof. Incertain embodiments, the immunomodulatory fusion protein comprises atleast one Fc domain which comprises at least one hinge domain or portionthereof and at least one CH2 domain or portion thereof (e.g., in thehinge-CH2 orientation). In certain embodiments, the immunomodulatoryfusion protein comprises at least one Fc domain which comprises at leastone CH2 domain or portion thereof and at least one CH3 domain or portionthereof (e.g., in the CH2-CH3 orientation). In certain embodiments, theimmunomodulatory fusion protein comprises at least one Fc domaincomprising at least one hinge domain or portion thereof, at least oneCH2 domain or portion thereof, and least one CH3 domain or portionthereof, for example in the orientation hinge-CH2-CH3, hinge-CH3-CH2, orCH2-CH3-hinge.

In certain embodiments, immunomodulatory fusion protein comprises atleast one complete Fc region derived from one or more immunoglobulinheavy chains (e.g., an Fc domain including hinge, CH2, and CH3 domains,although these need not be derived from the same antibody). In certainembodiments, immunomodulatory fusion protein comprises at least twocomplete Fc domains derived from one or more immunoglobulin heavychains. In certain embodiments, the complete Fc domain is derived from ahuman IgG immunoglobulin heavy chain (e.g., human IgG1).

In certain embodiments, the immunomodulatory fusion protein comprises atleast one Fc domain comprising a complete CH3 domain. In certainembodiments, the immunomodulatory fusion protein comprises at least oneFc domain comprising a complete CH2 domain. In certain embodiments, theimmunomodulatory fusion protein comprises at least one Fc domaincomprising at least a CH3 domain, and at least one of a hinge region,and a CH2 domain. In certain embodiments, the immunomodulatory fusionprotein comprises at least one Fc domain comprising a hinge and a CH3domain. In certain embodiments, the immunomodulatory fusion proteincomprises at least one Fc domain comprising a hinge, a CH2, and a CH3domain. In certain embodiments, the Fc domain is derived from a humanIgG immunoglobulin heavy chain (e.g., human IgG1).

The constant region domains or portions thereof making up an Fc domainof the immunomodulatory fusion protein may be derived from differentimmunoglobulin molecules. For example, a polypeptide suitable for use inthe immunomodulatory fusion proteins disclosed herein may comprise a CH2domain or portion thereof derived from an IgG1 molecule and a CH3 regionor portion thereof derived from an IgG3 molecule. In some embodiments,the immunomodulatory fusion protein comprises an Fc domain comprising ahinge domain derived, in part, from an IgG1 molecule and, in part, froman IgG3 molecule. As set forth herein, it will be understood by one ofordinary skill in the art that an Fc domain may be altered such that itvaries in amino acid sequence from a naturally occurring antibodymolecule.

In certain embodiments, the immunomodulatory fusion protein lacks one ormore constant region domains of a complete Fc region, i.e., they arepartially or entirely deleted. In certain embodiments, theimmunomodulatory fusion protein lacks an entire CH2 domain. In certainembodiments, the immunomodulatory fusion protein comprises CH2domain-deleted Fc regions derived from a vector (e.g., from IDECPharmaceuticals, San Diego) encoding an IgG1 human constant regiondomain (see, e.g., WO02/060955A2 and WO02/096948A2). This exemplaryvector is engineered to delete the CH2 domain and provide a syntheticvector expressing a domain-deleted IgG1 constant region. It will benoted that these exemplary constructs are preferably engineered to fusea binding CH3 domain directly to a hinge region of the respective Fcdomain.

In other constructs it may be desirable to provide a peptide spacerbetween one or more constituent Fc domains. For example, a peptidespacer may be placed between a hinge region and a CH2 domain and/orbetween a CH2 and a CH3 domain. For example, compatible constructs couldbe expressed wherein the CH2 domain has been deleted and the remainingCH3 domain (synthetic or unsynthetic) is joined to the hinge region witha 1-20, 1-10, or 1-5 amino acid peptide spacer. Such a peptide spacermay be added, for instance, to ensure that the regulatory elements ofthe constant region domain remain free and accessible or that the hingeregion remains flexible. Preferably, any linker peptide compatible usedin the instant disclosure will be relatively non-immunogenic and notprevent proper folding of the Fc.

In certain embodiments, an Fc domain employed in the immunomodulatoryfusion protein is altered or modified, e.g., by amino acid mutation(e.g., addition, deletion, or substitution). As used herein, the term“Fc domain variant” refers to an Fc domain having at least one aminoacid modification, such as an amino acid substitution, as compared tothe wild-type Fc from which the Fc domain is derived. For example,wherein the Fc domain is derived from a human IgG1 antibody, a variantcomprises at least one amino acid mutation (e.g., substitution) ascompared to a wild type amino acid at the corresponding position of thehuman IgG1 Fc region.

In certain embodiments, the Fc variant comprises a substitution at anamino acid position located in a hinge domain or portion thereof. Incertain embodiments, the Fc variant comprises a substitution at an aminoacid position located in a CH2 domain or portion thereof. In certainembodiments, the Fc variant comprises a substitution at an amino acidposition located in a CH3 domain or portion thereof. In certainembodiments, the Fc variant comprises a substitution at an amino acidposition located in a CH4 domain or portion thereof.

In certain embodiments, the immunomodulatory fusion protein comprises anFc variant comprising more than one amino acid substitution. Theimmunomodulatory fusion protein may comprise, for example, 2, 3, 4, 5,6, 7, 8, 9, 10 or more amino acid substitutions in the Fc domain.Preferably, the amino acid substitutions are spatially positioned fromeach other by an interval of at least 1 amino acid position or more, forexample, at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid positions ormore. More preferably, the engineered amino acids are spatiallypositioned apart from each other by an interval of at least 5, 10, 15,20, or 25 amino acid positions or more.

In some embodiments, an Fc domain includes changes in the region betweenamino acids 234-238, including the sequence LLGGP at the beginning ofthe CH2 domain. In some embodiments, an Fc variant alters Fc mediatedeffector function, particularly ADCC, and/or decrease binding avidityfor Fc receptors. In some aspects, sequence changes closer to theCH2-CH3 junction, at positions such as K322 or P331 can eliminatecomplement mediated cytotoxicity and/or alter avidity for FcR binding.In some embodiments, an Fc domain incorporates changes at residues P238and P331, e.g., changing the wild type prolines at these positions toserine. In some embodiments, alterations in the hinge region at one ormore of the three hinge cysteines, to encode CCC, SCC, SSC, SCS, or SSSat these residues can also affect FcR binding and molecular homogeneity,e.g., by elimination of unpaired cysteines that may destabilize thefolded protein.

Other amino acid mutations in the Fc domain are contemplated to reducebinding to the Fc gamma receptor and Fc gamma receptor subtypes. Forexample, mutations at positions 238, 239, 248, 249, 252, 254, 255, 256,258, 265, 267, 268, 269, 270, 272, 279, 280, 283, 285, 298, 289, 290,292, 293, 294, 295, 296, 298, 301, 303, 305, 307, 312, 315, 322, 324,327, 329, 330, 331, 333, 334, 335, 337, 338, 340, 356, 360, 373, 376,378, 379, 382, 388, 389, 398, 414, 416, 419, 430, 434, 435, 437, 438 or439 of the Fc region can alter binding as described in U.S. Pat. No.6,737,056, issued May 18, 2004, incorporated herein by reference in itsentirety. This patent reported that changing Pro331 in IgG3 to Serresulted in six fold lower affinity as compared to unmutated IgG3,indicating the involvement of Pro331 in Fc gamma RI binding. Inaddition, amino acid modifications at positions 234, 235, 236, and 237,297, 318, 320 and 322 are disclosed as potentially altering receptorbinding affinity in U.S. Pat. No. 5,624,821, issued Apr. 29, 1997 andincorporated herein by reference in its entirety.

Further mutations contemplated for use include, e.g., those described inU.S. Pat. App. Pub. No. 2006/0235208, published Oct. 19, 2006 andincorporated herein by reference in its entirety. Additionally,mutations described in U.S. Pat. App. Pub. No. 2006/0235208,incorporated herein by reference in its entirety, are contemplated foruse. The mutant L234A/L235A is described, e.g., in U.S. Pat. App. Pub.No. 2003/0108548, published Jun. 12, 2003 and incorporated herein byreference in its entirety. In embodiments, the described modificationsare included either individually or in combination. In certainembodiments, the mutation is D265A in human IgG1.

In certain embodiments, the immunomodulatory fusion protein comprises anFc variant comprising an amino acid substitution which alters theantigen-dependent effector functions of the polypeptide, in particularADCC or complement activation, e.g., as compared to a wild type Fcregion. Such immunomodulatory fusion protein exhibit decreased bindingto FcR gamma when compared to wild-type polypeptides and, therefore,mediate reduced effector function. Fc variants with decreased FcR gammabinding affinity are expected to reduce effector function, and suchmolecules are also useful, for example, for treatment of conditions inwhich target cell destruction is undesirable, e.g., where normal cellsmay express target molecules, or where chronic administration of thepolypeptide might result in unwanted immune system activation.

In certain embodiments, the immunomodulatory fusion protein exhibitsaltered binding to an activating FcγR (e.g. Fcγl, Fcγlla, or FcγRIIIa).In certain embodiments, the immunomodulatory fusion protein exhibitsaltered binding affinity to an inhibitory FcγR (e.g. FcγRIIb). Exemplaryamino acid substitutions which altered FcR or complement bindingactivity are disclosed in International PCT Publication No. WO05/063815which is incorporated by reference herein.

In some embodiments, the immunomodulatory fusion protein comprises anamino acid substitution which alters the glycosylation of the fusionprotein. For example, in some embodiments, the Fc domain comprises amutation leading to reduced glycosylation (e.g., N- or O-linkedglycosylation) or comprises an altered glycoform of the wild-type Fcdomain (e.g., a low fucose or fucose-free glycan). In certainembodiments, the immunomodulatory fusion protein has an amino acidsubstitution near or within a glycosylation motif, for example, anN-linked glycosylation motif that contains the amino acid sequence NXTor NXS. Exemplary amino acid substitutions which reduce or alterglycosylation are disclosed in WO05/018572 and US2007/0111281, thecontents of which are incorporated by reference herein. In certainembodiments, the immunomodulatory fusion protein comprises at least oneFc domain having engineered cysteine residue or analog thereof which islocated at the solvent-exposed surface. In certain embodiments, theimmunomodulatory fusion protein comprise an Fc domain comprising atleast one engineered free cysteine residue or analog thereof that issubstantially free of disulfide bonding with a second cysteine residue.Any of the above engineered cysteine residues or analogs thereof maysubsequently be conjugated to a functional domain using art-recognizedtechniques (e.g., conjugated with a thiol-reactive heterobifunctionallinker).

In certain embodiments, the immunomodulatory fusion protein comprises agenetically fused Fc domain having two or more of its constituent Fcdomains independently selected from the Fc domains described herein. Incertain embodiments, the Fc domains are the same. In certainembodiments, at least two of the Fc domains are different. For example,the Fc domains comprise the same number of amino acid residues or theymay differ in length by one or more amino acid residues (e.g., by about5 amino acid residues (e.g., 1, 2, 3, 4, or 5 amino acid residues),about 10 residues, about 15 residues, about 20 residues, about 30residues, about 40 residues, or about 50 residues). In certainembodiments, the Fc domains differ in sequence at one or more amino acidpositions. For example, at least two of the Fc domains may differ atabout 5 amino acid positions (e.g., 1, 2, 3, 4, or 5 amino acidpositions), about 10 positions, about 15 positions, about 20 positions,about 30 positions, about 40 positions, or about 50 positions).

C. Additional Linkers

In some embodiments, the linker suitable for use in the immunomodulatoryfusion protein described herein is a polyethylene glycol (PEG) domain.PEG is a well-known, water soluble polymer that is commerciallyavailable or can be prepared by ring-opening polymerization of ethyleneglycol according to methods well known in the art (Sandler and Karo,Polymer Synthesis, Academic Press, New York, Vol. 3, pages 138-161). Theterm “PEG” is used broadly to encompass any polyethylene glycolmolecule, and can be represented by the formula:X-0(CH₂CH₂0)_(n-1)CH₂CH₂OH, where n is 20 to 2300 and X is H or aterminal modification, e.g., a C₁₋₄ alkyl. In certain embodiments, thePEG suitable for use in the methods disclosed herein terminates on oneend with hydroxy or methoxy, i.e., X is H or CH₃ (“methoxy PEG”). PEGcan contain further chemical groups which are necessary for bindingreactions; which results from the chemical synthesis of the molecule; orwhich is a spacer for optimal distance of parts of the molecule. Inaddition, such a PEG can consist of one or more PEG side-chains whichare linked together. PEGs with more than one PEG chain are calledmultiarmed or branched PEGs. Branched PEGs can be prepared, for example,by the addition of polyethylene oxide to various polyols, includingglycerol, pentaerythriol, and sorbitol. For example, a four-armedbranched PEG can be prepared from pentaerythriol and ethylene oxide.Branched PEG are described in, for example, EP-A 0 473 084 and U.S. Pat.No. 5,932,462, both of which are hereby incorporated by reference. Oneform of PEGs includes two PEG side-chains (PEG2) linked via the primaryamino groups of a lysine (Monfardini et al., Bioconjugate Chem 1995;6:62-9).

In certain embodiments, PEG is conjugated to a cysteine moiety at the N-or C-terminus of the domains of the immunomodulatory fusion protein(e.g., immunomodulatory domain and collagen-binding domain). A PEGmoiety may also be attached by other chemistry, including by conjugationto amines. PEG conjugation to peptides or proteins generally involvesthe activation of PEG and coupling of the activated PEG-intermediatesdirectly to target proteins/peptides or to a linker, which issubsequently activated and coupled to target proteins/peptides (seeAbuchowski et al., JBC 1977; 252:3571 and JBC 1977; 252:3582, and Harriset. al., in: Poly(ethylene glycol) Chemistry: Biotechnical andBiomedical Applications; (J. M. Harris ed.) Plenum Press: New York,1992; Chap. 21 and 22). A variety of molecular mass forms of PEG can beselected, e.g., from about 1,000 Daltons (Da) to 100,000 Da (n is 20 to2300). The number of repeating units “n” in the PEG is approximated forthe molecular mass described in Daltons.

One skilled in the art can select a suitable molecular mass for PEG,e.g., based on at least the molecular mass of the immunomodulatoryfusion protein without PEG.

In certain embodiments, PEG molecules may be activated to react withamino groups on the domains such as with lysines (Bencham C. O. et al.,Anal. Biochem., 131, 25 (1983); Veronese, F. M. et al., Appl. Biochem.,11, 141 (1985); Zalipsky, S. et al., Polymeric Drugs and Drug DeliverySystems, adrs 9-110 ACS Symposium Series 469 (1999); Zalipsky, S. etal., Europ. Polym. J., 19, 1177-1183 (1983); Delgado, C. et al.,Biotechnology and Applied Biochemistry, 12, 119-128 (1990)).

In certain embodiments, carbonate esters of PEG are used to conjugatePEG. N,N′-disuccinimidylcarbonate (DSC) may be used in the reaction withPEG to form active mixed PEG-succinimidyl carbonate that may besubsequently reacted with a nucleophilic group of a linker or an aminogroup of IL-2 (see U.S. Pat. Nos. 5,281,698 and 5,932,462). In a similartype of reaction, 1,1′-(dibenzotriazolyl)carbonate anddi-(2-pyridyl)carbonate may be reacted with PEG to formPEG-benzotriazolyl and PEG-pyridyl mixed carbonate (U.S. Pat. No.5,382,657), respectively. Pegylation can be performed according to themethods of the state of the art, for example by reaction of IL-2 withelectrophilically active PEGs (Shearwater Corp., USA,www.shearwatercorp.com). Preferred PEG reagents suitable for use in themethods disclosed herein are, e.g., N-hydroxysuccinimidyl propionates(PEG-SPA), butanoates (PEG-SBA), PEG-succinimidyl propionate or branchedN-hydroxysuccinimides such as mPEG2-NHS (Monfardini, C, et al.,Bioconjugate Chem. 6 (1995) 62-69).

In some embodiments, the linker suitable for use in the immunomodulatoryfusion protein described herein is transferrin, as disclosed in U.S.Pat. Nos. 7,176,278 and 8,158,579, which are herein incorporated byreference in their entirety.

In some embodiments, the linker suitable for use in the immunomodulatoryfusion protein described herein is a serum immunoglobulin bindingprotein such as those disclosed in US2007/0178082, which is hereinincorporated by reference in its entirety.

In some embodiments, the linker suitable for use in the immunomodulatoryfusion protein described herein is a globulin such as thyroxine-bindingglobulin, a2 macroglobulin, or haptoglobulin.

In some embodiments, the linker suitable for use in the immunomodulatoryfusion protein described herein is a fibronectin (Fn)-based scaffolddomain protein, such as those disclosed in US2012/0094909, which isherein incorporated by reference in its entirety. Methods of makingfibronectin-based scaffold domain proteins are also disclosed inUS2012/0094909. A non-limiting example of an Fn3-based extended-PK groupis Fn3(HSA).

D. Other Linkers

In some embodiments, the immunomodulatory domain is operably linked to acollagen-binding domain via a linker, e.g., a gly-ser linker. In someembodiments, the immunomodulatory domain is operably linked to acollagen-binding domain via a linker (e.g., serum albumin), wherein thelinker is linked to the collagen-binding domain and immunomodulatorydomain via additional linkers (e.g. gly-ser linker). Linkers suitablefor fusing the collagen-binding domain and immunomodulatory domain, orfor fusing the collagen-binding domain, the immunomodulatory domain, andthe linker (e.g., serum albumin) are well known in the art, and aredisclosed in, e.g., US2010/0210511 US2010/0179094, and US2012/0094909,which are herein incorporated by reference in its entirety. Exemplarylinkers include gly-ser polypeptide linkers, glycine-proline polypeptidelinkers, and proline-alanine polypeptide linkers. In certainembodiments, the linker is a gly-ser polypeptide linker, i.e., a peptidethat consists of glycine and serine residues.

Exemplary gly-ser polypeptide linkers comprise the amino acid sequenceSer(Gly₄Ser)n. In certain embodiments, n=1. In certain embodiments, n=2.In certain embodiments, n=3, i.e., Ser(Gly₄Ser)3. In certainembodiments, n=4, i.e., Ser(Gly₄Ser)4. In certain embodiments, n=5. Incertain embodiments, n=6. In certain embodiments, n=7. In certainembodiments, n=8. In certain embodiments, n=9. In certain embodiments,n=10. Another exemplary gly-ser polypeptide linker comprises the aminoacid sequence Ser(Gly₄Ser)n. In certain embodiments, n=1. In certainembodiments, n=2. In certain embodiments, n=3. In certain embodiments,n=4. In certain embodiments, n=5. In certain embodiments, n=6. Anotherexemplary gly-ser polypeptide linker comprises (Gly₄Ser)n. In certainembodiments, n=1. In certain embodiments, n=2. In certain embodiments,n=3. In certain embodiments, n=4. In certain embodiments, n=5. Incertain embodiments, n=6. Another exemplary gly-ser polypeptide linkercomprises (Gly₃Ser)n. In certain embodiments, n=1. In certainembodiments, n=2. In certain embodiments, n=3. In certain embodiments,n=4. In certain embodiments, n=5. In certain embodiments n=6.

Other linkers that are suitable for use in the immunomodulatory fusionproteins are known in the art, for example, the serine-rich linkersdisclosed in U.S. Pat. No. 5,525,491, the helix forming peptide linkers(e.g., A(EAAAK)nA (n=2-5))(SEQ ID NO: 200) disclosed in Arai et al.,Protein Eng 2001; 14:529-32, and the stable linkers disclosed in Chen etal., Mol Pharm 2011; 8:457-65, i.e., the dipeptide linker LE, athrombin-sensitive disulfide cyclopeptide linker, and the alpha-helixforming linker LEA(EAAAK)₄ALEA(EAAAK)₄ALE (SEQ ID NO: 119).

Other exemplary linkers include GS linkers (i.e., (GS)n), GGSG (SEQ IDNO: 201) linkers (i.e., (GGSG)n)(SEQ ID NO: 202), GSAT linkers (SEQ IDNO: 203), SEG linkers, and GGS linkers (i.e., (GGSGGS)n)(SEQ ID NO:204), wherein n is a positive integer (e.g., 1, 2, 3, 4, or 5). Othersuitable linkers for use in the hybrid nuclease-albumin molecules can befound using publicly available databases, such as the Linker Database(ibi.vu.nl/programs/linkerdbwww). The Linker Database is a database ofinter-domain linkers in multi-functional enzymes which serve aspotential linkers in novel fusion proteins (see, e.g., George et al.,Protein Engineering 2002; 15:871-9).

It will be understood that variant forms of these exemplary polypeptidelinkers can be created by introducing one or more nucleotidesubstitutions, additions or deletions into the nucleotide sequenceencoding a polypeptide linker such that one or more amino acidsubstitutions, additions or deletions are introduced into thepolypeptide linker. Mutations may be introduced by standard techniques,such as site-directed mutagenesis and PCR-mediated mutagenesis.

Polypeptide linkers of the disclosure are at least one amino acid inlength and can be of varying lengths. In one embodiment, a polypeptidelinker of the disclosure is from about 1 to about 50 amino acids inlength. As used in this context, the term “about” indicates +/− twoamino acid residues. Since linker length must be a positive integer, thelength of from about 1 to about 50 amino acids in length, means a lengthof from 1 to 48-52 amino acids in length. In another embodiment, apolypeptide linker of the disclosure is from about 10-20 amino acids inlength. In another embodiment, a polypeptide linker of the disclosure isfrom about 15 to about 50 amino acids in length.

In another embodiment, a polypeptide linker of the disclosure is fromabout 20 to about 45 amino acids in length. In another embodiment, apolypeptide linker of the disclosure is from about 15 to about 25 aminoacids in length. In another embodiment, a polypeptide linker of thedisclosure is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35,36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53,54, 55, 56, 57, 58, 59, 60, or 61 or more amino acids in length.

Polypeptide linkers can be introduced into polypeptide sequences usingtechniques known in the art. Modifications can be confirmed by DNAsequence analysis. Plasmid DNA can be used to transform host cells forstable production of the polypeptides produced.

IV. Exemplary Immunomodulatory Fusion Proteins

The disclosure provides immunomodulatory fusion proteins comprising animmunomodulatory domain and a collagen-binding domain, optionally alinker, wherein the immunomodulatory domain is operably linked with orwithout the linker to the collagen-binding domain. The immunomodulatoryfusion proteins of the disclosure are modular and can be configured toincorporate various individual domains.

A. IL-2 Fusion Proteins

In some embodiments, the immunomodulatory fusion protein comprises IL-2and lumican, wherein IL-2 is operably linked to lumican. In someembodiments, IL-2 is operably linked to lumican with albumin. In someembodiments, IL-2 is operably linked to the N-terminus of lumican. Insome embodiments, IL-2 is operably linked to the C-terminus of lumican.

In some embodiments, the immunomodulatory fusion protein comprises thehuman IL-2 sequence set forth in SEQ ID NO: 1 operably linked to thehuman lumican sequence set forth in SEQ ID NO: 107. In some embodiments,the immunomodulatory fusion protein comprises the human IL-2 sequenceset forth in SEQ ID NO: 1 operably linked to the human lumican sequenceset forth in SEQ ID NO: 107 with a human serum albumin sequence selectedfrom SEQ ID NO: 42 and SEQ ID NO: 43.

In some embodiments, the immunomodulatory fusion protein comprises IL-2and LAIR-1, wherein IL-2 is operably linked to LAIR-1. In someembodiments, IL-2 is operably linked to LAIR-1 with albumin. In someembodiments, IL-2 is operably linked to the N-terminus of LAIR-1. Insome embodiments, IL-2 is operably linked to the C-terminus of LAIR-1.

In some embodiments, the immunomodulatory fusion protein comprises thehuman IL-2 sequence set forth in SEQ ID NO: 1 operably linked to thehuman LAIR-1 sequence set forth in SEQ ID NO: 98. In some embodiments,the immunomodulatory fusion protein comprises the human IL-2 sequenceset forth in SEQ ID NO: 1 operably linked to the human LAIR-1 sequenceset forth in SEQ ID NO: 98 with a human serum albumin sequence selectedfrom SEQ ID NO: 42 and SEQ ID NO: 43.

B. IL-12 Fusion Proteins

In some embodiments, the immunomodulatory fusion protein comprises IL-12and lumican, wherein IL-12 is operably linked to lumican. In someembodiments, IL-12 is operably linked to lumican with albumin. In someembodiments, IL-12 is operably linked to the N-terminus of lumican. Insome embodiments, IL-12 is operably linked to the C-terminus of lumican.

In some embodiments, the immunomodulatory fusion protein comprises thehuman IL-12 sequences set forth in SEQ ID NO: 2 and SEQ ID NO: 3operably linked to the human lumican sequence set forth in SEQ ID NO:107. In some embodiments, the immunomodulatory fusion protein comprisesthe human IL-12 sequences set forth in SEQ ID NO: 2 and SEQ ID NO: 3operably linked to the human lumican sequence set forth in SEQ ID NO:107 with a human serum albumin sequence selected from SEQ ID NO: 42 andSEQ ID NO: 43.

In some embodiments, the immunomodulatory fusion protein comprises IL-12and LAIR-1, wherein IL-12 is operably linked to LAIR-1. In someembodiments, IL-12 is operably linked to LAIR-1 with albumin. In someembodiments, IL-12 is operably linked to the N-terminus of LAIR-1. Insome embodiments, IL-12 is operably linked to the C-terminus of LAIR-1.

In some embodiments, the immunomodulatory fusion protein comprises thehuman IL-12 sequences set forth in SEQ ID NO: 2 and SEQ ID NO: 3,operably linked to the human LAIR-1 sequence set forth in SEQ ID NO: 98.In some embodiments, the immunomodulatory fusion protein comprises thehuman IL-12 sequences set forth in SEQ ID NO: 2 and SEQ ID NO: 3operably linked to the human LAIR-1 sequence set forth in SEQ ID NO: 98with a human serum albumin sequence selected from SEQ ID NO: 42 and SEQID NO: 43.

C. CCL-3 Fusion Proteins

In some embodiments, the immunomodulatory fusion protein comprises CCL-3and lumican, wherein CCL-3 is operably linked to lumican. In someembodiments, CCL-3 is operably linked to lumican with albumin. In someembodiments, CCL-3 is operably linked to the N-terminus of lumican. Insome embodiments, CCL-3 is operably linked to the C-terminus of lumican.

In some embodiments, the immunomodulatory fusion protein comprises thehuman CCL-3 sequence set forth in SEQ ID NO: 41 operably linked to thehuman lumican sequence set forth in SEQ ID NO: 107. In some embodiments,the immunomodulatory fusion protein comprises the human CCL-3 sequenceset forth in SEQ ID NO: 41 operably linked to the human lumican sequenceset forth in SEQ ID NO: 107 with a human serum albumin sequence selectedfrom SEQ ID NO: 42 and SEQ ID NO: 43.

In some embodiments, the immunomodulatory fusion protein comprises CCL-3and LAIR-1, wherein CCL-3 is operably linked to LAIR-1. In someembodiments, CCL-3 is operably linked to LAIR-1 with albumin. In someembodiments, CCL-3 is operably linked to the N-terminus of LAIR-1. Insome embodiments, CCL-3 is operably linked to the C-terminus of LAIR-1.

In some embodiments, the immunomodulatory fusion protein comprises thehuman CCL-3 sequence set forth in SEQ ID NO: 41 operably linked to thehuman LAIR-1 sequence set forth in SEQ ID NO: 98. In some embodiments,the immunomodulatory fusion protein comprises the human CCL-3 sequenceset forth in SEQ ID NO: 41 operably linked to the human LAIR-1 sequenceset forth in SEQ ID NO: 98 with a human serum albumin sequence selectedfrom SEQ ID NO: 42 and SEQ ID NO: 43.

D. CCL-4 Fusion Proteins

In some embodiments, the immunomodulatory fusion protein comprises CCL-4and lumican, wherein CCL-4 is operably linked to lumican. In someembodiments, CCL-4 is operably linked to lumican with albumin. In someembodiments, CCL-4 is operably linked to the N-terminus of lumican. Insome embodiments, CCL-4 is operably linked to the C-terminus of lumican.

In some embodiments, the immunomodulatory fusion protein comprises thehuman CCL-4 sequence set forth in SEQ ID NO: 33 operably linked to thehuman lumican sequence set forth in SEQ ID NO: 107. In some embodiments,the immunomodulatory fusion protein comprises the human CCL-4 sequenceset forth in SEQ ID NO: 33 operably linked to the human lumican sequenceset forth in SEQ ID NO: 107 with a human serum albumin sequence selectedfrom SEQ ID NO: 42 and SEQ ID NO: 43.

In some embodiments, the immunomodulatory fusion protein comprises CCL-4and LAIR-1, wherein CCL-4 is operably linked to LAIR-1. In someembodiments, CCL-4 is operably linked to LAIR-1 with albumin. In someembodiments, CCL-4 is operably linked to the N-terminus of LAIR-1. Insome embodiments, CCL-4 is operably linked to the C-terminus of LAIR-1.

In some embodiments, the immunomodulatory fusion protein comprises thehuman CCL-4 sequence set forth in SEQ ID NO: 33 operably linked to thehuman LAIR-1 sequence set forth in SEQ ID NO: 98. In some embodiments,the immunomodulatory fusion protein comprises the human CCL-4 sequenceset forth in SEQ ID NO: 33 operably linked to the human LAIR-1 sequenceset forth in SEQ ID NO: 98 with a human serum albumin sequence selectedfrom SEQ ID NO: 42 and SEQ ID NO: 43.

E. CCL-5 Fusion Proteins

In some embodiments, the immunomodulatory fusion protein comprises CCL-5and lumican, wherein CCL-5 is operably linked to lumican. In someembodiments, CCL-5 is operably linked to lumican with albumin. In someembodiments, CCL-5 is operably linked to the N-terminus of lumican. Insome embodiments, CCL-5 is operably linked to the C-terminus of lumican.

In some embodiments, the immunomodulatory fusion protein comprises thehuman CCL-5 sequence set forth in SEQ ID NO: 39 operably linked to thehuman lumican sequence set forth in SEQ ID NO: 107. In some embodiments,the immunomodulatory fusion protein comprises the human CCL-5 sequenceset forth in SEQ ID NO: 39 operably linked to the human lumican sequenceset forth in SEQ ID NO: 107 with a human serum albumin sequence selectedfrom SEQ ID NO: 42 and SEQ ID NO: 43.

In some embodiments, the immunomodulatory fusion protein comprises CCL-5and LAIR-1, wherein CCL-5 is operably linked to LAIR-1. In someembodiments, CCL-5 is operably linked to LAIR-1 with albumin. In someembodiments, CCL-5 is operably linked to the N-terminus of LAIR-1. Insome embodiments, CCL-5 is operably linked to the C-terminus of LAIR-1.

In some embodiments, the immunomodulatory fusion protein comprises thehuman CCL-5 sequence set forth in SEQ ID NO: 39 operably linked to thehuman LAIR-1 sequence set forth in SEQ ID NO: 98. In some embodiments,the immunomodulatory fusion protein comprises the human CCL-5 sequenceset forth in SEQ ID NO: 39 operably linked to the human LAIR-1 sequenceset forth in SEQ ID NO: 98 with a human serum albumin sequence selectedfrom SEQ ID NO: 42 and SEQ ID NO: 43.

F. Eotaxin Fusion Proteins

In some embodiments, the immunomodulatory fusion protein comprisesEotaxin and lumican, wherein Eotaxin is operably linked to lumican. Insome embodiments, Eotaxin is operably linked to lumican with albumin. Insome embodiments, Eotaxin is operably linked to the N-terminus oflumican. In some embodiments, Eotaxin is operably linked to theC-terminus of lumican.

In some embodiments, the immunomodulatory fusion protein comprises thehuman Eotaxin sequence set forth in SEQ ID NO: 38 operably linked to thehuman lumican sequence set forth in SEQ ID NO: 107. In some embodiments,the immunomodulatory fusion protein comprises the human Eotaxin sequenceset forth in SEQ ID NO: 38 operably linked to the human lumican sequenceset forth in SEQ ID NO: 107 with a human serum albumin sequence selectedfrom SEQ ID NO: 42 and SEQ ID NO: 43.

In some embodiments, the immunomodulatory fusion protein comprisesEotaxin and LAIR-1, wherein Eotaxin is operably linked to LAIR-1. Insome embodiments, Eotaxin is operably linked to LAIR-1 with albumin. Insome embodiments, Eotaxin is operably linked to the N-terminus ofLAIR-1. In some embodiments, Eotaxin is operably linked to theC-terminus of LAIR-1.

In some embodiments, the immunomodulatory fusion protein comprises thehuman Eotaxin sequence set forth in SEQ ID NO: 38 operably linked to thehuman LAIR-1 sequence set forth in SEQ ID NO: 98. In some embodiments,the immunomodulatory fusion protein comprises the human Eotaxin sequenceset forth in SEQ ID NO: 38 operably linked to the human LAIR-1 sequenceset forth in SEQ ID NO: 98 with a human serum albumin sequence selectedfrom SEQ ID NO: 42 and SEQ ID NO: 43.

G. Antibody Fusion Proteins

In some embodiments, the immunomodulatory fusion protein comprises ananti-CD3 antibody and lumican, wherein the anti-CD3 antibody is operablylinked to lumican. In some embodiments, the anti-CD3 antibody isoperably linked to the N-terminus of lumican. In some embodiments,anti-CD3 antibody is operably linked to the C-terminus of lumican.

In some embodiments, the immunomodulatory fusion protein comprises ananti-CD3 antibody and LAIR-1, wherein the anti-CD3 antibody is operablylinked to LAIR-1. In some embodiments, the anti-CD3 antibody is operablylinked to the N-terminus of LAIR-1. In some embodiments, the anti-CD3antibody is operably linked to the C-terminus of LAIR-1.

In some embodiments, the immunomodulatory fusion protein comprises ananti-4-1-BB antibody and lumican, wherein the anti-4-1-BB antibody isoperably linked to lumican. In some embodiments, the anti-4-1-BBantibody is operably linked to the N-terminus of lumican. In someembodiments, anti-4-1-BB antibody is operably linked to the C-terminusof lumican.

In some embodiments, the immunomodulatory fusion protein comprises ananti-4-1-BB antibody and LAIR-1, wherein the anti-4-1-BB antibody isoperably linked to LAIR-1. In some embodiments, the anti-4-1-BB antibodyis operably linked to the N-terminus of LAIR-1. In some embodiments, theanti-4-1-BB antibody is operably linked to the C-terminus of LAIR-1.

In some embodiments, the immunomodulatory fusion protein comprises ananti-CD40 antibody and lumican, wherein the anti-CD40 antibody isoperably linked to lumican. In some embodiments, the anti-CD40 antibodyis operably linked to the N-terminus of lumican. In some embodiments,anti-CD40 antibody is operably linked to the C-terminus of lumican.

In some embodiments, the immunomodulatory fusion protein comprises ananti-CD40 antibody and LAIR-1, wherein the anti-CD40 antibody isoperably linked to LAIR-1. In some embodiments, the anti-CD40 antibodyis operably linked to the N-terminus of LAIR-1. In some embodiments, theanti-CD40 antibody is operably linked to the C-terminus of LAIR-1.

In some embodiments, the immunomodulatory fusion protein comprises ananti-OX40 antibody and lumican, wherein the anti-OX40 antibody isoperably linked to lumican. In some embodiments, the anti-OX40 antibodyis operably linked to the N-terminus of lumican. In some embodiments,anti-OX40 antibody is operably linked to the C-terminus of lumican.

In some embodiments, the immunomodulatory fusion protein comprises ananti-OX40 antibody and LAIR-1, wherein the anti-OX40 antibody isoperably linked to LAIR-1. In some embodiments, the anti-OX40 antibodyis operably linked to the N-terminus of LAIR-1. In some embodiments, theanti-OX40 antibody is operably linked to the C-terminus of LAIR-1.

V. Methods for Making Immunomodulatory Fusion Proteins

In some aspects, the polypeptides described herein (e.g.,collagen-binding domains, cytokines, antibodies) are made in transformedhost cells using recombinant DNA techniques. To do so, a recombinant DNAmolecule coding for the peptide is prepared. Methods of preparing suchDNA molecules are well known in the art. For instance, sequences codingfor the peptides could be excised from DNA using suitable restrictionenzymes. Alternatively, the DNA molecule could be synthesized usingchemical synthesis techniques, such as the phosphoramidate method. Also,a combination of these techniques could be used.

The methods of making polypeptides also include a vector capable ofexpressing the peptides in an appropriate host. The vector comprises theDNA molecule that codes for the peptides operatively linked toappropriate expression control sequences. Methods of affecting thisoperative linking, either before or after the DNA molecule is insertedinto the vector, are well known. Expression control sequences includepromoters, activators, enhancers, operators, ribosomal nuclease domains,start signals, stop signals, cap signals, polyadenylation signals, andother signals involved with the control of transcription or translation.

The resulting vector having the DNA molecule thereon is used totransform an appropriate host. This transformation may be performedusing methods well known in the art.

Any of a large number of available and well-known host cells may besuitable for use in the methods disclosed herein. The selection of aparticular host is dependent upon a number of factors recognized by theart. These include, for example, compatibility with the chosenexpression vector, toxicity of the peptides encoded by the DNA molecule,rate of transformation, ease of recovery of the peptides, expressioncharacteristics, bio-safety and costs. A balance of these factors mustbe struck with the understanding that not all hosts may be equallyeffective for the expression of a particular DNA sequence. Within thesegeneral guidelines, useful microbial hosts include bacteria (such as E.coli sp.), yeast (such as Saccharomyces sp.) and other fungi, insects,plants, mammalian (including human) cells in culture, or other hostsknown in the art.

Next, the transformed host is cultured and purified. Host cells may becultured under conventional fermentation conditions so that the desiredcompounds are expressed. Such fermentation conditions are well known inthe art. Finally, the peptides are purified from culture by methods wellknown in the art.

The compounds may also be made by synthetic methods. For example, solidphase synthesis techniques may be used. Suitable techniques are wellknown in the art, and include those described in Merrifield (1973),Chem. Polypeptides, pp. 335-61 (Katsoyannis and Panayotis eds.);Merrifield (1963), J. Am. Chem. Soc. 85: 2149; Davis et al. (1985),Biochem. Intl. 10: 394-414; Stewart and Young (1969), Solid PhasePeptide Synthesis; U.S. Pat. No. 3,941,763; Finn et al. (1976), TheProteins (3rd ed.) 2: 105-253; and Erickson et al. (1976), The Proteins(3rd ed.) 2: 257-527. Solid phase synthesis is the preferred techniqueof making individual peptides since it is the most cost-effective methodof making small peptides. Compounds that contain derivatized peptides orwhich contain non-peptide groups may be synthesized by well-knownorganic chemistry techniques.

Other methods are of molecule expression/synthesis are generally knownin the art to one of ordinary skill.

The nucleic acid molecules described above can be contained within avector that is capable of directing their expression in, for example, acell that has been transduced with the vector. Accordingly, in additionto polypeptide mutants, expression vectors containing a nucleic acidmolecule encoding a mutant and cells transfected with these vectors areamong the certain embodiments.

Vectors suitable for use include T7-based vectors for use in bacteria(see, for example, Rosenberg et al., Gene 56: 125, 1987), the pMSXNDexpression vector for use in mammalian cells (Lee and Nathans, J. Biol.Chem. 263:3521, 1988), and baculovirus-derived vectors (for example theexpression vector pBacPAKS from Clontech, Palo Alto, Calif.) for use ininsect cells. The nucleic acid inserts, which encode the polypeptide ofinterest in such vectors, can be operably linked to a promoter, which isselected based on, for example, the cell type in which expression issought. For example, a T7 promoter can be used in bacteria, a polyhedrinpromoter can be used in insect cells, and a cytomegalovirus ormetallothionein promoter can be used in mammalian cells. Also, in thecase of higher eukaryotes, tissue-specific and cell type-specificpromoters are widely available. These promoters are so named for theirability to direct expression of a nucleic acid molecule in a giventissue or cell type within the body. Skilled artisans are well aware ofnumerous promoters and other regulatory elements which can be used todirect expression of nucleic acids.

In addition to sequences that facilitate transcription of the insertednucleic acid molecule, vectors can contain origins of replication, andother genes that encode a selectable marker. For example, theneomycin-resistance (neo^(r)) gene imparts G418 resistance to cells inwhich it is expressed, and thus permits phenotypic selection of thetransfected cells. Those of skill in the art can readily determinewhether a given regulatory element or selectable marker is suitable foruse in a particular experimental context.

Viral vectors that are suitable for use include, for example,retroviral, adenoviral, and adeno-associated vectors, herpes virus,simian virus 40 (SV40), and bovine papilloma virus vectors (see, forexample, Gluzman (Ed.), Eukaryotic Viral Vectors, CSH Laboratory Press,Cold Spring Harbor, N.Y.).

Prokaryotic or eukaryotic cells that contain and express a nucleic acidmolecule that encodes a polypeptide mutant are also suitable for use. Acell is a transfected cell, i.e., a cell into which a nucleic acidmolecule, for example a nucleic acid molecule encoding a mutantpolypeptide, has been introduced by means of recombinant DNA techniques.The progeny of such a cell are also considered suitable for use in themethods disclosed herein.

The precise components of the expression system are not critical. Forexample, a polypeptide mutant can be produced in a prokaryotic host,such as the bacterium E. coli, or in a eukaryotic host, such as aninsect cell (e.g., an Sf21 cell), or mammalian cells (e.g., COS cells,NIH 3T3 cells, or HeLa cells). These cells are available from manysources, including the American Type Culture Collection (Manassas, Va.).In selecting an expression system, it matters only that the componentsare compatible with one another. Artisans or ordinary skill are able tomake such a determination. Furthermore, if guidance is required inselecting an expression system, skilled artisans may consult Ausubel etal. (Current Protocols in Molecular Biology, John Wiley and Sons, NewYork, N.Y., 1993) and Pouwels et al. (Cloning Vectors: A LaboratoryManual, 1985 Suppl. 1987).

The expressed polypeptides can be purified from the expression systemusing routine biochemical procedures, and can be used, e.g., astherapeutic agents, as described herein.

Pharmaceutical Compositions and Modes of Administration

In certain embodiments, the disclosure provides for a pharmaceuticalcomposition comprising an immunomodulatory fusion protein with apharmaceutically acceptable diluent, carrier, solubilizer, emulsifier,preservative and/or adjuvant.

In certain embodiments, acceptable formulation materials preferably arenontoxic to recipients at the dosages and concentrations employed. Incertain embodiments, the formulation material(s) are for s.c. and/orI.V. administration. In certain embodiments, the formulation material(s)are for local administration, e.g., intratumoral administration. Incertain embodiments, the pharmaceutical composition can containformulation materials for modifying, maintaining or preserving, forexample, the pH, osmolality, viscosity, clarity, color, isotonicity,odor, sterility, stability, rate of dissolution or release, adsorptionor penetration of the composition. In certain embodiments, suitableformulation materials include, but are not limited to, amino acids (suchas glycine, glutamine, asparagine, arginine or lysine); antimicrobials;antioxidants (such as ascorbic acid, sodium sulfite or sodiumhydrogen-sulfite); buffers (such as borate, bicarbonate, Tris-HCl,citrates, phosphates or other organic acids); bulking agents (such asmannitol or glycine); chelating agents (such as ethylenediaminetetraacetic acid (EDTA)); complexing agents (such as caffeine,polyvinylpyrrolidone, beta-cyclodextrin orhydroxypropyl-beta-cyclodextrin); fillers; monosaccharides;disaccharides; and other carbohydrates (such as glucose, mannose ordextrins); proteins (such as serum albumin, gelatin or immunoglobulins);coloring, flavoring and diluting agents; emulsifying agents; hydrophilicpolymers (such as polyvinylpyrrolidone); low molecular weightpolypeptides; salt-forming counterions (such as sodium); preservatives(such as benzalkonium chloride, benzoic acid, salicylic acid,thimerosal, phenethyl alcohol, methylparaben, propylparaben,chlorhexidine, sorbic acid or hydrogen peroxide); solvents (such asglycerin, propylene glycol or polyethylene glycol); sugar alcohols (suchas mannitol or sorbitol); suspending agents; surfactants or wettingagents (such as pluronics, PEG, sorbitan esters, polysorbates such aspolysorbate 20, polysorbate 80, triton, tromethamine, lecithin,cholesterol, tyloxapal); stability enhancing agents (such as sucrose orsorbitol); tonicity enhancing agents (such as alkali metal halides,preferably sodium or potassium chloride, mannitol sorbitol); deliveryvehicles; diluents; excipients and/or pharmaceutical adjuvants.(Remington's Pharmaceutical Sciences, 18th Edition, A. R. Gennaro, ed.,Mack Publishing Company (1995). In certain embodiments, the formulationcomprises PBS; 20 mM NaOAC, pH 5.2, 50 mM NaCl; and/or 10 mM NAOAC, pH5.2, 9% Sucrose. In certain embodiments, the optimal pharmaceuticalcomposition will be determined by one skilled in the art depending upon,for example, the intended route of administration, delivery format anddesired dosage. See, for example, Remington's Pharmaceutical Sciences,supra. In certain embodiments, such compositions may influence thephysical state, stability, rate of in vivo release and rate of in vivoclearance of the immunomodulatory fusion protein.

In some embodiments, the formulations comprising an immunomodulatoryfusion protein described herein are 4° C. to 37° C. when administered toa subject.

In certain embodiments, the primary vehicle or carrier in apharmaceutical composition can be either aqueous or non-aqueous innature. For example, in certain embodiments, a suitable vehicle orcarrier can be water for injection, physiological saline solution orartificial cerebrospinal fluid, possibly supplemented with othermaterials common in compositions for parenteral administration. Incertain embodiments, the saline comprises isotonic phosphate-bufferedsaline. In certain embodiments, neutral buffered saline or saline mixedwith serum albumin are further exemplary vehicles. In certainembodiments, pharmaceutical compositions comprise Tris buffer of aboutpH 7.0-8.5, or acetate buffer of about pH 4.0-5.5, which can furtherinclude sorbitol or a suitable substitute therefore. In certainembodiments, a composition comprising an immunomodulatory fusion proteinis prepared for storage by mixing the selected composition having thedesired degree of purity with optional formulation agents (Remington'sPharmaceutical Sciences, supra) in the form of a lyophilized cake or anaqueous solution. Further, in certain embodiments, a compositioncomprising an immunomodulatory fusion protein is formulated as alyophilizate using appropriate excipients such as sucrose.

In certain embodiments, the pharmaceutical composition is selected forparenteral delivery. In certain embodiments, the compositions areselected for inhalation or for delivery through the digestive tract,such as orally. The preparation of such pharmaceutically acceptablecompositions is within the ability of one skilled in the art.

In certain embodiments, the formulation components are present inconcentrations that are acceptable to the site of administration. Incertain embodiments, buffers are used to maintain the composition atphysiological pH or at a slightly lower pH, typically within a pH rangeof from about 5 to about 8.

In certain embodiments, when parenteral administration is contemplated,a therapeutic composition is in the form of a pyrogen-free, parenterallyacceptable aqueous solution comprising an immunomodulatory fusionprotein, in a pharmaceutically acceptable vehicle. In certainembodiments, a vehicle for parenteral injection is sterile distilledwater in which the immunomodulatory fusion protein is formulated as asterile, isotonic solution, properly preserved. In certain embodiments,the preparation can involve the formulation of the desired molecule withan agent, such as injectable microspheres, bio-erodible particles,polymeric compounds (such as polylactic acid or polyglycolic acid),beads or liposomes, that can provide for the controlled or sustainedrelease of the product which can then be delivered via a depotinjection. In certain embodiments, hyaluronic acid can also be used, andcan have the effect of promoting sustained duration in the circulation.In certain embodiments, implantable drug delivery devices can be used tointroduce the desired molecule.

In certain embodiments, a pharmaceutical composition is formulated forinhalation. In certain embodiments, an immunomodulatory fusion proteinis formulated as a dry powder for inhalation. In certain embodiments, aninhalation solution comprising an immunomodulatory fusion protein isformulated with a propellant for aerosol delivery. In certainembodiments, solutions can be nebulized. Pulmonary administration isfurther described in PCT application No. PCT/US94/001875, whichdescribes pulmonary delivery of chemically modified proteins.

In certain embodiments, it is contemplated that formulations areadministered orally. In certain embodiments, an immunomodulatory fusionprotein administered in this fashion is formulated with or without thosecarriers customarily used in the compounding of solid dosage forms suchas tablets and capsules. In certain embodiments, a capsule is designedto release the active portion of the formulation at the point in thegastrointestinal tract when bioavailability is maximized andpre-systemic degradation is minimized. In certain embodiments, at leastone additional agent is included to facilitate absorption of theimmunomodulatory fusion protein. In certain embodiments, diluents,flavorings, low melting point waxes, vegetable oils, lubricants,suspending agents, tablet disintegrating agents, and binders can also beemployed.

In certain embodiments, a pharmaceutical composition comprises aneffective quantity of immunomodulatory fusion protein in a mixture withnon-toxic excipients which are suitable for the manufacture of tablets.In certain embodiments, by dissolving the tablets in sterile water, oranother appropriate vehicle, solutions are prepared in unit-dose form.In certain embodiments, suitable excipients include, but are not limitedto, inert diluents, such as calcium carbonate, sodium carbonate orbicarbonate, lactose, or calcium phosphate; or binding agents, such asstarch, gelatin, or acacia; or lubricating agents such as magnesiumstearate, stearic acid, or talc.

Additional pharmaceutical compositions will be evident to those skilledin the art, including formulations involving an immunomodulatory fusionprotein, in sustained- or controlled-delivery formulations. In certainembodiments, techniques for formulating a variety of other sustained- orcontrolled-delivery means, such as liposome carriers, bio-erodiblemicroparticles or porous beads and depot injections, are also known tothose skilled in the art. See for example, PCT Application No.PCT/US93/00829 which describes the controlled release of porouspolymeric microparticles for the delivery of pharmaceuticalcompositions. In certain embodiments, sustained-release preparations caninclude semipermeable polymer matrices in the form of shaped articles,e.g. films, or microcapsules. Sustained release matrices can includepolyesters, hydrogels, polylactides (U.S. Pat. No. 3,773,919 and EP058,481), copolymers of L-glutamic acid and gamma ethyl-L-glutamate(Sidman et al., Biopolymers, 22:547-556 (1983)), poly(2-hydroxyethyl-methacrylate) (Langer et al., J. Biomed. Mater. Res.,15: 167-277 (1981) and Langer, Chem. Tech., 12:98-105 (1982)), ethylenevinyl acetate (Langer et al., supra) or poly-D(−)-3-hydroxybutyric acid(EP 133,988). In certain embodiments, sustained release compositionsinclude liposomes, which can be prepared by any of several methods knownin the art. See, e.g., Eppstein et al, Proc. Natl. Acad. Sci. USA,82:3688-3692 (1985); EP 036,676; EP 088,046 and EP 143,949.

The pharmaceutical composition to be used for in vivo administrationtypically is sterile. In certain embodiments, this is accomplished byfiltration through sterile filtration membranes. In certain embodiments,where the composition is lyophilized, sterilization using this method isconducted either prior to or following lyophilization andreconstitution. In certain embodiments, the composition for parenteraladministration can be stored in lyophilized form or in a solution. Incertain embodiments, parenteral compositions generally are placed into acontainer having a sterile access port, for example, an intravenoussolution bag or vial having a stopper pierceable by a hypodermicinjection needle.

In certain embodiments, once the pharmaceutical composition has beenformulated, it can be stored in sterile vials as a solution, suspension,gel, emulsion, solid, or as a dehydrated or lyophilized powder. Incertain embodiments, such formulations can be stored either in aready-to-use form or in a form (e.g., lyophilized) that is reconstitutedprior to administration.

In certain embodiments, kits are provided for producing a single-doseadministration unit. In certain embodiments, the kit can contain both afirst container having a dried protein and a second container having anaqueous formulation. In certain embodiments, kits containing single andmulti-chambered pre-filled syringes (e.g., liquid syringes andlyosyringes) are included.

In certain embodiments, the effective amount of a pharmaceuticalcomposition comprising immunomodulatory fusion protein to be employedtherapeutically will depend, for example, upon the therapeutic contextand objectives. One skilled in the art will appreciate that theappropriate dosage levels for treatment, according to certainembodiments, will thus vary depending, in part, upon the moleculedelivered, the indication for which the immunomodulatory fusion proteinis being used, the route of administration, and the size (body weight,body surface or organ size) and/or condition (the age and generalhealth) of the patient. In certain embodiments, the clinician can titerthe dosage and modify the route of administration to obtain the optimaltherapeutic effect.

In certain embodiments, the frequency of dosing will take into accountthe pharmacokinetic parameters of the immunomodulatory fusion protein inthe formulation used. In certain embodiments, a clinician willadminister the composition until a dosage is reached that achieves thedesired effect. In certain embodiments, the composition can therefore beadministered as a single dose, or as two or more doses (which may or maynot contain the same amount of the desired molecule) over time, or as acontinuous infusion via an implantation device or catheter. Furtherrefinement of the appropriate dosage is routinely made by those ofordinary skill in the art and is within the ambit of tasks routinelyperformed by them. In certain embodiments, appropriate dosages can beascertained through use of appropriate dose-response data.

In certain embodiments, the route of administration of thepharmaceutical composition is in accord with known methods, e.g. orally,through injection by intravenous, intraperitoneal, intracerebral(intra-parenchymal), intracerebroventricular, intramuscular,subcutaneously, intraocular, intraarterial, intraportal, orintralesional routes; by sustained release systems or by implantationdevices. In certain embodiments, the compositions can be administered bybolus injection or continuously by infusion, or by implantation device.In certain embodiments, individual elements of the combination therapymay be administered by different routes.

In certain embodiments, the composition can be administered locally viaimplantation of a membrane, sponge or another appropriate material ontowhich the desired molecule has been absorbed or encapsulated. In certainembodiments, where an implantation device is used, the device can beimplanted into any suitable tissue or organ, and delivery of the desiredmolecule can be via diffusion, timed-release bolus, or continuousadministration. In certain embodiments, it can be desirable to use apharmaceutical composition comprising an immunomodulatory fusion proteinin an ex vivo manner. In such instances, cells, tissues and/or organsthat have been removed from the patient are exposed to a pharmaceuticalcomposition comprising the immunomodulatory fusion protein after whichthe cells, tissues and/or organs are subsequently implanted back intothe patient.

In certain embodiments, an immunomodulatory fusion protein is deliveredby implanting certain cells that have been genetically engineered, usingmethods such as those described herein, to express and secrete thepolypeptides. In certain embodiments, such cells can be animal or humancells, and can be autologous, heterologous, or xenogeneic. In certainembodiments, the cells can be immortalized. In certain embodiments, inorder to decrease the chance of an immunological response, the cells canbe encapsulated to avoid infiltration of surrounding tissues. In certainembodiments, the encapsulation materials are typically biocompatible,semipermeable polymeric enclosures or membranes that allow the releaseof the protein product(s) but prevent the destruction of the cells bythe patient's immune system or by other detrimental factors from thesurrounding tissues.

Methods of Treating

The immunomodulatory fusion proteins and/or nucleic acids expressingthem, described herein, are useful for treating a disorder associatedwith abnormal apoptosis or a differentiative process (e.g., cellularproliferative disorders (e.g., hyperproliferaetive disorders) orcellular differentiative disorders, such as cancer). Non-limitingexamples of cancers that are amenable to treatment with the methods ofthe present disclosure are described below.

Examples of cellular proliferative and/or differentiative disordersinclude cancer (e.g., carcinoma, sarcoma, metastatic disorders orhematopoietic neoplastic disorders, e.g., leukemias). A metastatic tumorcan arise from a multitude of primary tumor types, including but notlimited to those of prostate, colon, lung, breast and liver.Accordingly, the compositions used herein, comprising, e.g.,immunomodulatory fusion protein, can be administered to a patient whohas cancer.

As used herein, the terms “cancer” (or “cancerous”),“hyperproliferative,” and “neoplastic” refer to cells having thecapacity for autonomous growth (i.e., an abnormal state or conditioncharacterized by rapidly proliferating cell growth). Hyperproliferativeand neoplastic disease states may be categorized as pathologic (i.e.,characterizing or constituting a disease state), or they may becategorized as non-pathologic (i.e., as a deviation from normal but notassociated with a disease state). The terms are meant to include alltypes of cancerous growths or oncogenic processes, metastatic tissues ormalignantly transformed cells, tissues, or organs, irrespective ofhistopathologic type or stage of invasiveness. “Pathologichyperproliferative” cells occur in disease states characterized bymalignant tumor growth. Examples of non-pathologic hyperproliferativecells include proliferation of cells associated with wound repair.

The terms “cancer” or “neoplasm” are used to refer to malignancies ofthe various organ systems, including those affecting the lung, breast,thyroid, lymph glands and lymphoid tissue, gastrointestinal organs, andthe genitourinary tract, as well as to adenocarcinomas which aregenerally considered to include malignancies such as most colon cancers,renal-cell carcinoma, prostate cancer and/or testicular tumors,non-small cell carcinoma of the lung, cancer of the small intestine andcancer of the esophagus.

The term “carcinoma” is art recognized and refers to malignancies ofepithelial or endocrine tissues including respiratory system carcinomas,gastrointestinal system carcinomas, genitourinary system carcinomas,testicular carcinomas, breast carcinomas, prostatic carcinomas,endocrine system carcinomas, and melanomas. The immunomodulatory fusionproteins can be used to treat patients who have, who are suspected ofhaving, or who may be at high risk for developing any type of cancer,including renal carcinoma or melanoma, or any viral disease. Exemplarycarcinomas include those forming from tissue of the cervix, lung,prostate, breast, head and neck, colon and ovary. The term also includescarcinosarcomas, which include malignant tumors composed ofcarcinomatous and sarcomatous tissues. An “adenocarcinoma” refers to acarcinoma derived from glandular tissue or in which the tumor cells formrecognizable glandular structures.

Additional examples of proliferative disorders include hematopoieticneoplastic disorders. As used herein, the term “hematopoietic neoplasticdisorders” includes diseases involving hyperplastic/neoplastic cells ofhematopoietic origin, e.g., arising from myeloid, lymphoid or erythroidlineages, or precursor cells thereof. Preferably, the diseases arisefrom poorly differentiated acute leukemias (e.g., erythroblasticleukemia and acute megakaryoblastic leukemia). Additional exemplarymyeloid disorders include, but are not limited to, acute promyeloidleukemia (APML), acute myelogenous leukemia (AML) and chronicmyelogenous leukemia (CML) (reviewed in Vaickus, L. (1991) Crit. Rev. inOncol./Hemotol. 11:267-97); lymphoid malignancies include, but are notlimited to acute lymphoblastic leukemia (ALL) which includes B-lineageALL and T-lineage ALL, chronic lymphocytic leukemia (CLL),prolymphocytic leukemia (PLL), hairy cell leukemia (HLL) andWaldenstrom's macro globulinemia (WM). Additional forms of malignantlymphomas include, but are not limited to non-Hodgkin lymphoma andvariants thereof, peripheral T cell lymphomas, adult T cellleukemia/lymphoma (ATL), cutaneous T cell lymphoma (CTCL), largegranular lymphocytic leukemia (LGF), Hodgkin's disease andReed-Sternberg disease.

It will be appreciated by those skilled in the art that amounts of animmunomodulatory fusion protein sufficient to reduce tumor growth andsize, or a therapeutically effective amount, will vary not only on theparticular compounds or compositions selected, but also with the routeof administration, the nature of the condition being treated, and theage and condition of the patient, and will ultimately be at thediscretion of the patient's physician or pharmacist. The length of timeduring which the compounds used in the instant method will be givenvaries on an individual basis.

It will be appreciated by those skilled in the art that the B16 melanomamodel used herein is a generalized model for solid tumors. That is,efficacy of treatments in this model is also predictive of efficacy ofthe treatments in other non-melanoma solid tumors. For example, asdescribed in Baird et al. (J Immunology 2013; 190:469-78; Epub Dec. 7,2012), efficacy of cps, a parasite strain that induces an adaptiveimmune response, in mediating anti-tumor immunity against B16F10 tumorswas found to be generalizable to other solid tumors, including models oflung carcinoma and ovarian cancer. In another example, results from aline of research into VEGF targeting lymphocytes also shows that resultsin B16F10 tumors were generalizable to the other tumor types studied(Chinnasamy et al., JCI 2010; 120:3953-68; Chinnasamy et al., ClinCancer Res 2012; 18:1672-83). In yet another example, immunotherapyinvolving LAG-3 and PD-1 led to reduced tumor burden, with generalizableresults in a fibrosarcoma and colon adenocarcinoma cell lines (Woo etal., Cancer Res 2012; 72:917-27).

In certain embodiments, the immunomodulatory fusion proteins disclosedherein are used to treat cancer. In certain embodiments, theimmunomodulatory fusion proteins disclosed herein are used to treatmelanoma, leukemia, lung cancer, breast cancer, prostate cancer, ovariancancer, colon cancer, and brain cancer.

In certain embodiments, the immunomodulatory fusion proteins disclosedherein inhibit the growth and/or proliferation of tumor cells. Incertain embodiments, the immunomodulatory fusion proteins disclosedherein reduce tumor size. In certain embodiments, the immunomodulatoryfusion proteins disclosed herein inhibit metastases of a primary tumor.

It will be appreciated by those skilled in the art that reference hereinto treatment extends to prophylaxis as well as the treatment of thenoted cancers and symptoms.

Combination Therapy

In some embodiments, the immunomodulatory fusion proteins are used incombination with other therapies. For example, in some embodiments theimmunomodulatory fusion proteins are used in combination with anotherimmunotherapy. Exemplary immunotherapies include, but are not limitedto, chimeric antigen receptor (CAR) T cell therapy, tumor-associatedantigen targeting antibodies, immune checkpoint inhibitors, and cancervaccines.

I. Chimeric Antigen Receptor (CAR) Effector Cells

In some aspects, the disclosure provides immunomodulatory fusionproteins to be used or performed in conjunction with chimeric antigenreceptor (CAR) effector cell therapy (e.g., CAR T cells).

Chimeric antigen receptors (CARs) are genetically-engineered, artificialtransmembrane receptors, which confer an arbitrary specificity for aligand onto an immune effector cell (e.g. a T cell, natural killer cellor other immune cell) and which results in activation of the effectorcell upon recognition and binding to the ligand. Typically thesereceptors are used to impart the antigen specificity of a monoclonalantibody onto a T cell.

In some embodiments, CARs contain three domains: 1) an ectodomaintypically comprising a signal peptide, a ligand or antigen recognitionregion (e.g. scFv), and a flexible spacer; 2) a transmembrane (TM)domain; 3) an endodomain (alternatively known as an “activation domain”)typically comprising one or more intracellular signaling domains. Theectodomain of the CAR resides outside of the cell and is exposed to theextracellular space, whereby it is accessible for interaction with itscognate ligand. The TM domain allows the CAR to be anchored into thecell membrane of the effector cell. The third endodomain (also known asthe “activation domain”) aids in effector cell activation upon bindingof the CAR to its specific ligand. In some embodiments, effector cellactivation comprises induction of cytokine and chemokine production, aswell as activation of the cytolytic activity of the cells. In someembodiments, the CARs redirect cytotoxicity toward tumor cells.

In some embodiments, CARs comprise a ligand- or antigen-specificrecognition domain that binds to a specific target ligand or antigen(also referred to as a binding domain). In some embodiments, the bindingdomain is a single-chain antibody variable fragment (scFv), a tetheredligand or the extracellular domain of a co-receptor, fused to atransmembrane domain, which is linked, in turn, to a signaling domain.In some embodiments, the signaling domain is derived from CD3 ζ or FcRγ.In some embodiments, the CAR comprises one or more co-stimulatorydomains derived from a protein such as CD28, CD137 (also known as4-1BB), CD134 (also known as OX40) and CD278 (also known as ICOS).

Engagement of the antigen binding domain of the CAR with its targetantigen on the surface of a target cell results in clustering of the CARand delivers an activation stimulus to the CAR-containing cell. In someembodiments, the main characteristic of CARs are their ability toredirect immune effector cell specificity, thereby triggeringproliferation, cytokine production, phagocytosis or production ofmolecules that can mediate cell death of the target antigen expressingcell in a major histocompatibility (MHC) independent manner, exploitingthe cell specific targeting abilities of monoclonal antibodies, solubleligands or cell specific co-receptors. Although scFv-based CARsengineered to contain a signaling domain from CD3 ζ or FcRγ have beenshown to deliver a potent signal for T cell activation and effectorfunction, they are not sufficient to elicit signals that promote T cellsurvival and expansion in the absence of a concomitant co-stimulatorysignal. A new generation of CARs containing a binding domain, a hinge, atransmembrane and the signaling domain derived from CD3 ζ or FcRγtogether with one or more co-stimulatory signaling domains (e.g.,intracellular co-stimulatory domains derived from CD28, CD137, CD134 andCD278) has been shown to more effectively direct antitumor activity aswell as increased cytokine secretion, lytic activity, survival andproliferation in CAR expressing T cells in vitro, in animal models andcancer patients (Milone et al., Molecular Therapy, 2009; 17: 1453-1464;Zhong et al., Molecular Therapy, 2010; 18: 413-420; Carpenito et al.,PNAS, 2009; 106:3360-3365).

In some embodiments, chimeric antigen receptor-expressing effector cells(e.g. CAR-T cells) are cells that are derived from a patient with adisease or condition and genetically modified in vitro to express atleast one CAR with an arbitrary specificity to a ligand. The cellsperform at least one effector function (e.g. induction of cytokines)that is stimulated or induced by the specific binding of the ligand tothe CAR and that is useful for treatment of the same patient's diseaseor condition. The effector cells may be T cells (e.g. cytotoxic T cellsor helper T cells). One skilled in the art would understand that othercell types (e.g. a natural killer cell or a stem cell) may express CARsand that a chimeric antigen receptor effector cell may comprise aneffector cell other than a T cell. In some embodiments, the effectorcell is a T cell (e.g. a cytotoxic T cell) that exerts its effectorfunction (e.g. a cytotoxic T cell response) on a target cell whenbrought in contact or in proximity to the target or target cell (e.g. acancer cell) (see e.g., Chang and Chen (2017) Trends Mol Med23(5):430-450).

Prolonged exposure of T cells to their cognate antigen can result inexhaustion of effector functions, enabling the persistence of infectedor transformed cells. Recently developed strategies to stimulate orrejuvenate host effector function using agents that induce an immunecheckpoint blockade have resulted in success towards the treatment ofseveral cancers. Emerging evidence suggests that T cell exhaustion mayalso represent a significant impediment in sustaining long-livedantitumor activity by chimeric antigen receptor-expressing T cells(CAR-T cells. In some embodiments, the differentiation status of thepatient-harvested T cells prior to CAR transduction and the conditioningregimen a patient undergoes before reintroducing the CAR-T cells (e.g.,addition or exclusion of alkylating agents, fludarabine, total-bodyirradiation) can profoundly affect the persistence and cytotoxicpotential of CAR-T cells. In vitro culture conditions that stimulate(via anti-CD3/CD28 or stimulator cells) and expand (via cytokines, suchas IL-2) T cell populations can also alter the differentiation statusand effector function of CAR-T cells (Ghoneim et al., (2016) Trends inMolecular Medicine 22(12):1000-1011).

In some embodiments, in particular for the treatment of ALL and/or NHL,suitable CARs target CD19 or CD20. Non-limiting examples include CARscomprising a structure: (i) an anti-CD19 scFv, a CD8 H/TM domain, an4-1BB CS domain and a CD3ζ TCR signaling domain; (ii) an anti-CD19 scFv,a CD28 hinge and transmembrane domain, a CD28 co-stimulatory domain anda CD3ζ TCR signaling domain; and (iii) an anti-CD20 scFv, an IgG hingeand transmembrane domain, a CD28/4-1BB co-stimulatory domain and a CD3ζTCR signaling domain. In some embodiments, a CAR effector cell suitablefor combination with the combinations and methods disclosed hereintargets CD19 or CD20, including but not limited to Kymriah™(tisagenlecleucel; Novartis; formerly CTL019) and Yescarta™(axicabtagene ciloleucel; Kite Pharma).

A. Re-Targeted CAR T Cells

In some embodiments, the CAR-T therapy suitable for use in combinationwith the immunomodulatory fusion proteins is a re-targeted CAR-T cell.In some embodiments, effector cells (e.g., T cells) modified to expressa CAR which binds to a universal immune receptor, a tag, a switch or anFc region on an immunoglobulin are suitable for the methods describedherein.

In some embodiments, effector cells (e.g., T cells) are modified toexpress a universal immune receptor or UnivIR. One type of UnivIR is abiotin-binding immune receptor (BBIR) (see e.g., US Patent PublicationUS20140234348 A1 incorporated herein by reference in its entirety).Other examples of methods and compositions relating to universalchimeric receptors and/or effector cells expressing universal chimericreceptors are described in International Patent ApplicationsWO2016123122A1, WO2017143094A1, WO2013074916A1, US Patent ApplicationUS20160348073A1, all of which are incorporated herein by reference intheir entirety.

In some embodiments, effector cells (e.g., T cells) are modified toexpress a universal, modular, anti-tag chimeric antigen receptor(UniCAR). This system allows for retargeting of UniCAR engrafted immunecells against multiple antigens (see e.g., US Patent PublicationUS20170240612 A1 incorporated herein by reference in its entirety;Cartellieri et al., (2016) Blood Cancer Journal 6, e458 incorporatedherein by reference in its entirety).

In some embodiments, effector cells (e.g., T cells) are modified toexpress a switchable chimeric antigen receptor and chimeric antigenreceptor effector cell (CAR-EC) switches. In this system, the CAR-ECswitches have a first region that is bound by a chimeric antigenreceptor on the CAR-EC and a second region that binds a cell surfacemolecule on target cell, thereby stimulating an immune response from theCAR-EC that is cytotoxic to the bound target cell. In some embodiments,the CAR-EC is a T cell, wherein the CAR-EC switch may act as an“on-switch” for CAR-EC activity. Activity may be “turned off” byreducing or ceasing administration of the switch. These CAR-EC switchesmay be used with CAR-ECs disclosed herein, as well as existing CART-cells, for the treatment of a disease or condition, such as cancer,wherein the target cell is a malignant cell. Such treatment may bereferred to herein as switchable immunotherapy (US Patent PublicationU.S. Pat. No. 9,624,276 B2 incorporated herein by reference in itsentirety). In some embodiments, effector cells (e.g., T cells) aremodified to express a receptor that binds the Fc portion of humanimmunoglobulins (e.g., CD16V-BB-ζ) (Kudo et al., (2014) Cancer Res74(1):93-103 incorporated herein by reference in its entirety).

In some embodiments, effector cells (e.g., T cells) are modified toexpress a universal immune receptor (e.g., switchable CAR, sCAR) thatbinds a peptide neo-epitope (PNE). In some embodiments, the peptideneo-epitope (PNE), has been incorporated at defined different locationswithin an antibody targeting an antigen (antibody switch). Therefore,sCAR-T-cell specificity is redirected only against PNE, not occurring inthe human proteome, thus allowing an orthogonal interaction between thesCAR-T-cell and the antibody switch. In this way, sCAR-T cells arestrictly dependent on the presence of the antibody switch to becomefully activated, thus excluding CAR T-cell off-target recognition ofendogenous tissues or antigens in the absence of the antibody switch(Arcangeli et al., (2016) Transl Cancer Res 5 (Suppl 2):S174-S177incorporated herein by reference in its entirety). Other examples ofswitchable CARs is provided by US Patent Application US20160272718A1incorporated herein by reference in its entirety.

As used herein, the term “tag” encompasses a universal immune receptor,a tag, a switch, or an Fc region of an immunoglobulin as describedsupra. In some embodiments, an effector cell is modified to express aCAR comprising a tag binding domain. In some embodiments, the CAR bindsfluorescein isothiocyanate (FITC), streptavidin, biotin, dinitrophenol,peridinin chlorophyll protein complex, green fluorescent protein,phycoerythrin (PE), horse radish peroxidase, palmitoylation,nitrosylation, alkalanine phosphatase, glucose oxidase, or maltosebinding protein.

B. Anti-TAG Chimeric Antigen Receptors (AT-CAR)

In some embodiments, the CAR-T therapy suitable for use in combinationwith the immunomodulatory fusion proteins is an anti-tag CAR T cell.There are several limitations to the generalized clinical application ofCAR T cells. For example, as there is no single tumor antigenuniversally expressed by all cancer types, each scFv in a CAR needs tobe engineered with specificity for the desired tumor antigen. Inaddition, tumor antigens targeted by a CAR may be down-regulated ormutated in response to treatment resulting in tumor evasion.

As an alternative, universal, anti-tag chimeric antigen receptors(AT-CAR) and CAR-T cells have been developed. For example, human T cellshave been engineered to express an anti-fluorescein isothiocyanate(FITC) CAR (referred to anti-FITC-CAR). This platform takes advantage ofthe high affinity interaction between the anti-FITC scFv (on the cell'ssurface) and FITC as well as the ability conjugate FITC molecules (orother tags) to any anti-cancer-based monoclonal antibody such ascetuximab (anti-EGFR), retuximab (anti-CD20) and herceptin (anti-Her2).

Accordingly, in some embodiments, effector cells (e.g., T cells) aremodified to express a universal anti-tag chimeric antigen receptor(AT-CAR), as described at least in WO 2012082841 and US20160129109A1,incorporated herein by reference in its entirety. In such AT-CARsystems, T cells recognize and bind tagged proteins, such as antibodies.For example, in some embodiments an AT-CAR T cell recognizes tag-labeledantibodies, such as FITC-labeled antibodies. In some embodiments, ananti-tumor antigen antibody is conjugated to a tag (e.g., FITC), andadministered prior to, concurrently, or after AT-CAR therapy. Anti-tumorantigen antibodies are known to those of skill in the art.

As indicated, the binding specificity of the tag-binding domain dependson the identity of the tag that is conjugated to the protein that isused to bind target cells. For example, in some aspects of thedisclosure, the tag is FITC, the tag-binding domain is an anti-FITCscFv. Alternatively, in some aspects of the disclosure, the tag isbiotin or PE (phycoerythrin) and the tag-binding domain is ananti-biotin scFv or an anti-PE scFv.

In some embodiments, the protein of each formulation of tagged proteinsis the same or different and the protein is an antibody or anantigen-binding fragment thereof. In some aspects, the antibody orantigen-binding fragment thereof is cetuximab (anti-EGFR), nimotuzumab(anti-EGFR), panitumumab (anti-EGFR), retuximab (anti-CD20), omalizumab(anti-CD20), tositumomab (anti-CD20), trastuzumab (anti-Her2),gemtuzumab (anti-CD33), alemtuzumab (anti-CD52), and bevacuzimab(anti-VEGF).

Thus, in some embodiments, the tagged proteins include FITC-conjugatedantibodies, biotin-conjugated antibodies, PE-conjugated antibodies,histidine-conjugated antibodies and streptavidin-conjugated antibodies,where the antibody binds to a TAA or a TSA expressed by the targetcells. For example, the tagged proteins include, but are not limited to,FITC-conjugated cetuximab, FITC-conjugated retuximab, FITC-conjugatedherceptin, biotin-conjugated cetuximab, biotin-conjugated retuximab,biotin-conjugated herceptin, PE-conjugated cetuximab, PE-conjugatedretuximab, PE-conjugated herceptin, histidine-conjugated cetuximab,histidine-conjugated retuximab, histidine-conjugated herceptin,streptavidin-conjugated cetuximab, streptavidin-conjugated retuximab,and streptavidin-conjugated herceptin.

In some embodiments, the AT-CAR of each population of AT-CAR-expressingT cells is the same or different and the AT-CAR comprises a tag-bindingdomain, a transmembrane domain, and an activation domain. In someembodiments, the tag-binding domain is an antibody or an antigen-bindingfragment thereof. In some aspects, the tag-binding domain specificallybinds FITC, biotin, PE, histidine or streptavidin. In some embodimentsthe tag-binding domain is antigen-binding fragment and theantigen-binding fragment is a single chain variable fragment (scFv),such as a scFv that specifically binds FITC, biotin, PE, histidine orstreptavidin. In some embodiments the transmembrane domain is the hingeand transmembrane regions of the human CD8α chain. In some embodiments,the activation domain comprises one or more of the cytoplasmic region ofCD28, the cytoplasmic region of CD137 (41BB), OX40, HVEM, CD3ζ and FcRε.

In some embodiments, the tag of each formulation of tagged proteins isthe same or different and the tag is selected from the group consistingof fluorescein isothiocyanate (FITC), streptavidin, biotin, histidine,dinitrophenol, peridinin chlorophyll protein complex, green fluorescentprotein, phycoerythrin (PE), horse radish peroxidase, palmitoylation,nitrosylation, alkalanine phosphatase, glucose oxidase, and maltosebinding protein.

The tag may be conjugated to the proteins using techniques such aschemical coupling and chemical cross-linkers. Alternatively,polynucleotide vectors can be prepared that encode the tagged proteinsas fusion proteins. Cell lines can then be engineered to express thetagged proteins, and the tagged proteins can be isolated from culturemedia, purified and used in the methods disclosed herein.

In some embodiments, tagged proteins are administered to a subject priorto, or concurrent with, or after administration of the AT-CAR-expressingT cells. In some embodiments, the disclosure provide a method oftreating cancer in a subject, comprising: (a) administering aformulation of tagged proteins to a subject in need of treatment,wherein the tagged proteins bind a cancer cell in the subject, and (b)administering a therapeutically-effective population of anti-tagchimeric antigen receptor (AT-CAR)-expressing T cells to the subject,wherein the AT-CAR-expressing T cells bind the tagged proteins andinduce cancer cell death, thereby treating cancer in a subject.

C. Tandem CAR (TanCAR) Effector Cells

In some embodiments, the CAR-T therapy suitable for use in combinationwith the immunomodulatory fusion proteins is a tandem CAR effector cell.It has been observed that using a CAR approach for cancer treatment,tumor heterogeneity and immunoediting can cause escape from CARtreatment (Grupp et al., New Eng. J. Med (2013) 368:1509-1518). As analternative approach, bispecific CARs, known as tandem CARs or TanCARs,have been developed in an attempt to target multiple cancer specificmarkers simultaneously. In a TanCAR, the extracellular domain comprisestwo antigen binding specificities in tandem, joined by a linker. The twobinding specificities (scFvs) are thus both linked to a singletransmembrane portion: one scFv being juxtaposed to the membrane and theother being in a distal position. As an exemplary TanCAR, Grada et al.(Mol Ther Nucleic Acids (2013) 2, e105) describes a TanCAR whichincludes a CD19-specific scFv, followed by a Gly-Ser linker and aHER2-specific scFv. The HER2-scFv was in the juxta-membrane position,and the CD19-scFv in the distal position. The TanCAR was shown to inducedistinct T cell reactivity against each of the two tumor restrictedantigens.

Accordingly, some aspects of the disclosure relate to a tandem chimericantigen receptor that mediates bispecific activation and targeting of Tcells. Although the present disclosure refers to bispecificity for theCAR, in some aspects the CARs are able to target three, four, or moretumor antigens. Targeting multiple antigens using CAR T cells mayenhance T cell activation and/or offset tumor escape by antigen loss.TanCARs may also target multiple expressed antigens, target varioustumors using the same cellular product with a broad specificity, and/orprovide a better toxicity profile with a less intensely signaling CARachieving the same results due to multiple specificity.

In some embodiments, the disclosure provides a TanCAR that includes twotargeting domains. In some embodiments, the disclosure provides amultispecific TanCAR that includes three or more targeting domains. Inanother embodiment, the disclosure provides a first CAR and second CARat the cell surface, each CAR comprising an antigen-binding domain,wherein the antigen-binding domain of the first CAR binds to a firsttumor antigen (e.g., CD19, CD20, CD22, HER2) and the antigen-bindingdomain of the second CAR binds to another (different) tumor antigen.TanCARs are described in US20160303230A1 and US20170340705A1,incorporated herein by reference.

In some embodiments, the TanCAR of the disclosure targets two or moretumor antigens. Exemplary tumor antigens include one or more of CD19,CD20, CD22, k light chain, CD30, CD33, CD123, CD38, ROR1, ErbB2,ErbB3/4, EGFR vIII, carcinoembryonic antigen, EGP2, EGP40, mesothelin,TAG72, PSMA, NKG2D ligands, B7-H6, IL-13 receptor a 2, MUC1, MUC16, CA9,GD2, GD3, HMW-MAA, CD171, Lewis Y, G250/CALX, HLA-AI MAGE A1, HLA-A2NY-ESO-1, PSC1, folate receptor-α, CD44v7/8, 8H9, NCAM, VEGF receptors,5T4, Fetal AchR, NKG2D ligands, CD44v6, TEM1, and/or TEM8.

In some embodiments, the disclosure provides a bispecific TanCAR thattargets CD19 and another tumor antigen. In some embodiments, thedisclosure provides a bispecific TanCAR that targets CD22 and anothertumor antigen. In some embodiments, the disclosure provides a bispecificTanCAR that targets HER2 and another tumor antigen. In some embodiments,the disclosure provides a bispecific TanCAR that targets IL13R-alpha2and another tumor antigen.

In some embodiments, the disclosure provides a bispecific TanCAR thattargets VEGF-A and another tumor antigen. In some embodiments, thedisclosure provides a bispecific TanCAR that targets Tem8 and anothertumor antigen. In some embodiments, the disclosure provides a bispecificTanCAR that targets FAP and another tumor antigen. In some embodiments,the disclosure provides a bispecific TanCAR that targets EphA2 andanother tumor antigen. In some embodiments, the disclosure provides abispecific TanCAR that targets one or more, two or more, three or more,or four or more of the following tumor antigens: CD19, CD22, HER2,IL13R-alpha2, VEGF-A, Tem8, FAP, or EphA2, and any combination thereof.In some embodiments, the disclosure provides a bispecific TanCAR thattargets HER2 and IL13R-alpha2. In some embodiments, the disclosureprovides a bispecific TanCAR that targets CD19 and CD22.

D. Methods for Generating Chimeric Antigen Receptors and CAR EffectorCells

In some embodiments, a subject's effectors cells (e.g., T cells) aregenetically modified with a chimeric antigen receptor (Sadelain et al.,Cancer Discov. 3:388-398, 2013). For example, an effector cell (e.g., Tcell) is provided and a recombinant nucleic acid encoding a chimericantigen receptor is introduced into the patient-derived effector cell(e.g., T cell) to generate a CAR cell. In some embodiments, effectorcells (e.g., T cells) not derived from the subject are geneticallymodified with a chimeric antigen receptor. For example, in someembodiments, effector cells (e.g., T cells) are allogeneic cells thathave been engineered to be used as an “off the shelf” adoptive celltherapy, such as Universal Chimeric Antigen Receptor T cells (UCARTs),as developed by Cellectis. UCARTs are allogeneic CAR T cells that havebeen engineered to be used for treating the largest number of patientswith a particular cancer type. Non-limiting examples of UCARTs underdevelopment by Cellectis include those that target the following tumorantigens: CD19, CD123, CD22, CS1 and CD38.

A variety of different methods known in the art can be used to introduceany of the nucleic acids or expression vectors disclosed herein into aneffector cell (e.g., T cell). Non-limiting examples of methods forintroducing nucleic acid into a an effector cell (e.g., T cell) include:lipofection, transfection (e.g., calcium phosphate transfection,transfection using highly branched organic compounds, transfection usingcationic polymers, dendrimer-based transfection, optical transfection,particle-based transfection (e.g., nanoparticle transfection), ortransfection using liposomes (e.g., cationic liposomes)),microinjection, electroporation, cell squeezing, sonoporation,protoplast fusion, impalefection, hydrodynamic delivery, gene gun,magnetofection, viral transfection, and nucleofection. Furthermore, theCRISPR/Cas9 genome editing technology known in the art can be used tointroduce CAR nucleic acids into effector cells (e.g., T cells) and/orto introduce other genetic modifications (e.g., as described below) intoeffector cells (e.g., T cells) to enhance CAR cell activity (for use ofCRISPR/Cas9 technology in connection with CAR T cells, see e.g., U.S.Pat. Nos. 9,890,393; 9,855,297; US 2017/0175128; US 2016/0184362; US2016/0272999; WO 2015/161276; WO 2014/191128; CN 106755088; CN106591363; CN 106480097; CN 106399375; CN 104894068).

Provided herein are methods that can be used to generate any of thecells or compositions described herein where each cell can express a CAR(e.g., any of the CARs described herein).

Chimeric antigen receptors (CARs) include an antigen-binding domain, atransmembrane domain, and an cytoplasmic signaling domain that includesa cytoplasmic sequence of CD3ζ sequence sufficient to stimulate a T cellwhen the antigen-binding domain binds to the antigen, and optionally, acytoplasmic sequence of one or more (e.g., two, three, or four)co-stimulatory proteins (e.g., a cytoplasmic sequence of one or more ofB7-H3, BTLA, CD2, CD7, CD27, CD28, CD30, CD40, CD40L, CD80, CD160,CD244, ICOS, LAG3, LFA-1, LIGHT, NKG2C, 4-1BB, OX40, PD-1, PD-L, TIM3,and a ligand that specifically binds to CD83) that provides forco-stimulation of the T cell when the antigen-binding domain binds tothe antigen. In some embodiments, a CAR can further include a linker.Non-limiting aspects and features of CARs are described below.Additional aspects of CARs and CAR cells, including exemplaryantigen-binding domains, linkers, transmembrane domains, and cytoplasmicsignaling domains, are described in, e.g., Kakarla et al., Cancer J.20:151-155, 2014; Srivastava et al., Trends Immunol. 36:494-502, 2015;Nishio et al., Oncoimmunology 4(2): e988098, 2015; Ghorashian et al.,Br. J. Haematol. 169:463-478, 2015; Levine, Cancer Gene Ther. 22:79-84,2015; Jensen et al., Curr. Opin. Immunol. 33:9-15, 2015; Singh et al.,Cancer Gene Ther. 22:95-100, 2015; Li et al., Zhongguo Shi Yan Xue YeXue Za Zhi 22:1753-1756, 2014; Gill et al., Immunol. Rev. 263:68-89,2015; Magee et al., Discov. Med. 18:265-271, 2014; Gargett et al.,Front. Pharmacol. 5:235, 2014; Yuan et al., Zhongguo Shi Yan Xue Ye XueZa Zhi 22:1137-1141, 2014; Pedgram et al., Cancer J. 20:127-133, 2014;Eshhar et al., Cancer J. 20:123-126, 2014; Ramos et al., Cancer J.20:112-118, 2014; Maus et al., Blood 123:2625-2635, 2014; Jena et al.,Curr. Hematol. Malig. Rep. 9:50-56, 2014; Maher et al., Curr. Gene Ther.14:35-43, 2014; Riches et al., Discov. Med. 16:295-302, 2013; Cheadle etal., Immunol. Rev. 257:83-90, 2014; Davila et al., Int. J. Hematol.99:361-371, 2014; Xu et al., Cancer Lett. 343:172-178, 2014;Kochenderfer et al., Nat. Rev. Clin. Oncol. 10:267-276, 2013; Hosing etal., Curr. Hematol. Malig. Rep. 8:60-70, 2013; Hombach et al., Curr.Mol. Med. 13:1079-1088, 2013; Xu et al., Leuk. Lymphoma 54:255-260,2013; Gilham et al., Trends Mol. Med. 18:377-384, 2012; Lipowska-Bhallaet al., Cancer Immunol. Immunother. 61:953-962, 2012; Chmielewski etal., Cancer Immunol. Immunother. 61:1269-1277, 2013; Jena et al., Blood116:1035-1044, 2010; Dotti et al, Immunology Reviews 257(1): 107-126,2013; Dai et al., Journal of the National Cancer Institute 108(7):djv439, 2016; Wang and Riviere, Molecular Therapy-Oncolytics 3: 16015,2016; U.S. Patent Application Publication Nos. 2018/0057609;2018/0037625; 2017/0362295; 2017/0137783; 2016/0152723, 2016/0206656,2016/0199412, 2016/0208018, 2015/0232880, 2015/0225480; 2015/0224143;2015/0224142; 2015/0190428; 2015/0196599; 2015/0152181; 2015/0140023;2015/0118202; 2015/0110760; 2015/0099299; 2015/0093822; 2015/0093401;2015/0051266; 2015/0050729; 2015/0024482; 2015/0023937; 2015/0017141;2015/0017136; 2015/0017120; 2014/0370045; 2014/0370017; 2014/0369977;2014/0349402; 2014/0328812; 2014/0322275; 2014/0322216; 2014/0322212;2014/0322183; 2014/0314795; 2014/0308259; 2014/0301993; 2014/0296492;2014/0294784; 2014/0286973; 2014/0274909; 2014/0274801; 2014/0271635;2014/0271582; 2014/0271581; 2014/0271579; 2014/0255363; 2014/0242701;2014/0242049; 2014/0227272; 2014/0219975; 2014/0170114; 2014/0134720;2014/0134142; 2014/0120622; 2014/0120136; 2014/0106449; 2014/0106449;2014/0099340; 2014/0086828; 2014/0065629; 2014/0050708; 2014/0024809;2013/0344039; 2013/0323214; 2013/0315884; 2013/0309258; 2013/0288368;2013/0287752; 2013/0287748; 2013/0280221; 2013/0280220; 2013/0266551;2013/0216528; 2013/0202622; 2013/0071414; 2012/0321667; 2012/0302466;2012/0301448; 2012/0301447; 2012/0060230; 2011/0213288; 2011/0158957;2011/0104128; 2011/0038836; 2007/0036773; and 2004/0043401. Additionalaspects of CARs and CAR cells, including exemplary antigen-bindingdomains, linkers, transmembrane domains, and cytoplasmic signalingdomains, are described in WO 2016/168595; WO 12/079000; 2015/0141347;2015/0031624; 2015/0030597; 2014/0378389; 2014/0219978; 2014/0206620;2014/0037628; 2013/0274203; 2013/0225668; 2013/0116167; 2012/0230962;2012/0213783; 2012/0093842; 2012/0071420; 2012/0015888; 2011/0268754;2010/0297093; 2010/0158881; 2010/0034834; 2010/0015113; 2009/0304657;2004/0043401; 2014/0322253; 2015/0118208; 2015/0038684; 2014/0024601;2012/0148552; 2011/0223129; 2009/0257994; 2008/0160607; 2008/0003683;2013/0121960; 2011/0052554; and 2010/0178276.

Antigen Binding Domains

Antigen binding domains included in the chimeric antigen receptor (CAR)can specifically bind to an antigen (e.g., a tumor associated antigen(TAA) or an antigen that is not expressed on a non-cancerous cell) or auniversal receptor (e.g., a tag). Non-limiting examples of an antigenbinding domain include: a monoclonal antibody (e.g., IgG1, IgG2, IgG3,IgG4, IgM, IgE, and IgD) (e.g., a fully human or a chimeric (e.g., ahumanized) antibody), an antigen binding fragment of an antibody (e.g.,Fab, Fab′, or F(ab′)₂ fragments) (e.g., a fragment of a fully human or achimeric (e.g., humanized) antibody), a diabody, a triabody, atetrabody, a minibody, a scFv, scFv-Fc, (scFv)₂, scFab, bis-scFv,hc-IgG, a BiTE, a single domain antibody (e.g., a V-NAR domain or a VhHdomain), IgNAR, and a multispecific (e.g., bispecific antibody)antibody. Methods of making these antigen-binding domains are known inthe art.

In some embodiments, an antigen binding domain includes at least one(e.g., one, two, three, four, five, or six) CDR (e.g., any of the threeCDRs from an immunoglobulin light chain variable domain or any of thethree CDRs from an immunoglobulin heavy chain variable domain) of anantibody that is capable of specifically binding to the target antigen,such as immunoglobulin molecules (e.g., light or heavy chainimmunoglobulin molecules) and immunologically-active (antigen-binding)fragments of immunoglobulin molecules.

In some embodiments, an antigen binding domain is a single-chainantibody (e.g., a V-NAR domain or a V_(H)H domain, or any of thesingle-chain antibodies as described herein). In some embodiments, anantigen binding domain is a whole antibody molecule (e.g., a human,humanized, or chimeric antibody) or a multimeric antibody (e.g., abi-specific antibody).

In some embodiments, antigen-binding domains include antibody fragmentsand multispecific (e.g., bi-specific) antibodies or antibody fragments.Examples of antibodies and antigen-binding fragments thereof include,but are not limited to: single-chain Fvs (scFvs), Fab fragments, Fab′fragments, F(ab′)₂, disulfide-linked Fvs (sdFvs), Fvs, and fragmentscontaining either a VL or a VH domain.

Additional antigen binding domains provided herein are polyclonal,monoclonal, multispecific (multimeric, e.g., bi-specific), humanantibodies, chimeric antibodies (e.g., human-mouse chimera),single-chain antibodies, intracellularly-made antibodies (i.e.,intrabodies), and antigen-binding fragments thereof. The antibodies orantigen-binding fragments thereof can be of any type (e.g., IgG, IgE,IgM, IgD, IgA, and IgY), class (e.g., IgG₁, IgG₂, IgG₃, IgG₄, IgA₁, andIgA₂), or subclass. In some embodiments, the antigen binding domain isan IgG₁ antibody or antigen-binding fragment thereof. In some examples,the antigen binding domain is an IgG₄ antibody or antigen-bindingfragment thereof. In some embodiments, the antigen binding domain is animmunoglobulin comprising a heavy and light chain.

Additional examples of antigen binding domains are antigen-bindingfragments of an IgG (e.g., an antigen-binding fragment of IgG1, IgG2,IgG3, or IgG4) (e.g., an antigen-binding fragment of a human orhumanized IgG, e.g., human or humanized IgG1, IgG2, IgG3, or IgG4), anantigen-binding fragment of an IgA (e.g., an antigen-binding fragment ofIgA1 or IgA2) (e.g., an antigen-binding fragment of a human or humanizedIgA, e.g., a human or humanized IgA1 or IgA2), an antigen-bindingfragment of an IgD (e.g., an antigen-binding fragment of a human orhumanized IgD), an antigen-binding fragment of an IgE (e.g., anantigen-binding fragment of a human or humanized IgE), or anantigen-binding fragment of an IgM (e.g., an antigen-binding fragment ofa human or humanized IgM).

In some embodiments, an antigen binding domain can bind to a particularantigen (e.g., a tumor-associated antigen) with an affinity (K_(D))about or less than 1×10⁻⁷ M (e.g., about or less than 1×10⁻⁸ M, about orless than 5×10⁻⁹ M, about or less than 2×10⁻⁹ M, or about or less than1×10⁻⁹ M), e.g., in saline or in phosphate buffered saline.

In some embodiments, CAR effector cells (e.g., CAR T cells) comprise aCAR molecule that binds to a tumor antigen (e.g., comprises a tumorantigen binding domain). In some embodiments, the CAR molecule comprisesan antigen binding domain that recognizes a tumor antigen of a solidtumor (e.g., breast cancer, colon cancer, etc.). In some embodiments,the CAR molecule is a tandem CAR molecule as described supra, whichcomprises at least two antigen binding domains. In some embodiments, theCAR molecule comprises an antigen binding domain that recognizes a tumorantigen of a hematologic malignancy (e.g., leukemia, acute lymphocyticleukemia, acute myelocytic leukemia, acute promyelocytic leukemia,chronic leukemia, chronic myelocytic (granulocytic) leukemia, chroniclymphocytic leukemia, mantle cell lymphoma, primary central nervoussystem lymphoma, Burkitt's lymphoma and marginal zone B cell lymphoma,Polycythemia vera, Hodgkin's disease, non-Hodgkin's disease, multiplemyeloma, etc.).

In some embodiments, the tumor antigen is a tumor-specific antigen(TSA). A TSA is unique to tumor cells and does not occur on other cellsin the body. In some embodiments, the tumor antigen is atumor-associated antigen (TAA). A TAA is not unique to a tumor cell andinstead is also expressed on a normal cell under conditions that fail toinduce a state of immunologic tolerance to the antigen. The expressionof the antigen on the tumor may occur under conditions that enable theimmune system to respond to the antigen. In some embodiments, a TAA isexpressed on normal cells during fetal development when the immunesystem is immature and unable to respond or is normally present atextremely low levels on normal cells but which are expressed at muchhigher levels on tumor cells.

In certain embodiments, the tumor-associated antigen is determined bysequencing a patient's tumor cells and identifying mutated proteins onlyfound in the tumor. These antigens are referred to as “neoantigens.”Once a neoantigen has been identified, therapeutic antibodies can beproduced against it and used in the methods described herein.

In some embodiments, the tumor antigen is an epithelial cancer antigen,(e.g., breast, gastrointestinal, lung), a prostate specific cancerantigen (PSA) or prostate specific membrane antigen (PSMA), a bladdercancer antigen, a lung (e.g., small cell lung) cancer antigen, a coloncancer antigen, an ovarian cancer antigen, a brain cancer antigen, agastric cancer antigen, a renal cell carcinoma antigen, a pancreaticcancer antigen, a liver cancer antigen, an esophageal cancer antigen, ahead and neck cancer antigen, or a colorectal cancer antigen. In certainembodiments, the tumor antigen is a lymphoma antigen (e.g.,non-Hodgkin's lymphoma or Hodgkin's lymphoma), a B-cell lymphoma cancerantigen, a leukemia antigen, a myeloma (e.g., multiple myeloma or plasmacell myeloma) antigen, an acute lymphoblastic leukemia antigen, achronic myeloid leukemia antigen, or an acute myelogenous leukemiaantigen.

Tumor antigens, (e.g. tumor-associated antigens (TAAs) andtumor-specific antigens (TSAs)) that may be targeted by CAR effectorcells (e.g., CAR T cells), include, but are not limited to, 1GH-IGK,43-9F, 5T4, 791Tgp72, acyclophilin C-associated protein,alphafetoprotein (AFP), α-actinin-4, A3, antigen specific for A33antibody, ART-4, B7, Ba 733, BAGE, BCR-ABL, beta-catenin, beta-HCG,BrE3-antigen, BCA225, BTAA, CA125, CA 15-3\CA 27.29\BCAA, CA195, CA242,CA-50, CAM43, CAMEL, CAP-1, carbonic anhydrase IX, c-Met, CA19-9,CA72-4, CAM 17.1, CASP-8/m, CCCL19, CCCL21, CD1, CD1a, CD2, CD3, CD4,CD5, CD8, CD11A, CD14, CD15, CD16, CD18, CD19, CD20, CD21, CD22, CD23,CD25, CD29, CD30, CD32b, CD33, CD37, CD38, CD40, CD40L, CD44, CD45,CD46, CD52, CD54, CD55, CD59, CD64, CD66a-e, CD67, CD68, CD70, CD70L,CD74, CD79a, CD79b, CD80, CD83, CD95, CD126, CD132, CD133, CD138, CD147,CD154, CDC27, CDK4, CDK4m, CDKN2A, CO-029, CTLA4, CXCR4, CXCR7, CXCL12,HIF-1a, colon-specific antigen-p (CSAp), CEA (CEACAM5), CEACAM6, c-Met,DAM, E2A-PRL, EGFR, EGFRvIII, EGP-1 (TROP-2), EGP-2, ELF2-M, Ep-CAM,fibroblast growth factor (FGF), FGF-5, Flt-1, Flt-3, folate receptor,G250 antigen, Ga733VEpCAM, GAGE, gp100, GRO-β, H4-RET, HLA-DR, HM1.24,human chorionic gonadotropin (HCG) and its subunits, HER2/neu, HMGB-1,hypoxia inducible factor (HIF-1), HSP70-2M, HST-2, HTgp-175, Ia, IGF-1R,IFN-γ, IFN-α, IFN-β, IFN-λ, IL-4R, IL-6R, IL-13R, IL-15R, IL-3R,IL-117R, IL-18R, IL-2, IL-6, IL-8, IL-12, IL-15, IL-17, IL-18, IL-23,IL-25, insulin-like growth factor-1 (IGF-1), KC4-antigen, KSA,KS-1-antigen, KS1-4, LAGE-1a, Le-Y, LDR/FUT, M344, MA-50, macrophagemigration inhibitory factor (MIF), MAGE, MAGE-1, MAGE-3, MAGE-4, MAGE-5,MAGE-6, MART-1, MART-2, TRAG-3, mCRP, MCP-1, MIP-1A, MIP-1B, MIF,MG7-Ag, MOV18, MUC1, MUC2, MUC3, MUC4, MUC5ac, MUC13, MUC16, MUM-1/2,MUM-3, MYL-RAR, NB/70K, Nm23H1, NuMA, NCA66, NCA95, NCA90, NY-ESO-1,p15, p16, p185erbB2, p180erbB3, PAM4 antigen, pancreatic cancer mucin,PD1 receptor (PD-1), PD-1 receptor ligand 1 (PD-L1), PD-1 receptorligand 2 (PD-L2), PI5, placental growth factor, p53, PLAGL2, Pmel17prostatic acid phosphatase, PSA, PRAME, PSMA, PlGF, ILGF, ILGF-1R, IL-6,IL-25, RCAS1, RS5, RAGE, RANTES, Ras, T101, SAGE, S100, survivin,survivin-2B, SDDCAG16, TA-90\Mac2 binding protein, TAAL6, TAC, TAG-72,TLP, tenascin, TRAIL receptors, TRP-1, TRP-2, TSP-180, TNF-α, Tnantigen, Thomson-Friedenreich antigens, tumor necrosis antigens,tyrosinase, VEGFR, ED-B fibronectin, WT-1, 17-1A-antigen, complementfactors C3, C3a, C3b, C5a, C5, an angiogenesis marker, bcl-2, bcl-6, andK-ras, an oncogene marker and an oncogene product (see, e.g., Sensi etal., Clin Cancer Res 2006, 12:5023-32; Parmiani et al., J Immunol 2007,178:1975-79; Novellino et al. Cancer Immunol Immunother 2005,54:187-207).

In some embodiments, the tumor antigen is a viral antigen derived from avirus associated with a human chronic disease or cancer (such ascervical cancer). For example, in some embodiments, the viral antigen isderived from Epstein-Barr virus (EBV), HPV antigens E6 and/or E7,hepatitis C virus (HCV), hepatitis B virus (HBV), or cytomegalovirus(CMV).

Exemplary cancers or tumors and specific tumor antigens associated withsuch tumors (but not exclusively), include acute lymphoblastic leukemia(etv6, aml1, cyclophilin b), B cell lymphoma (Ig-idiotype), glioma(E-cadherin, α-catenin, β-catenin, γ-catenin, p120ctn), bladder cancer(p21ras), biliary cancer (p21ras), breast cancer (MUC family, HER2/neu,c-erbB-2), cervical carcinoma (p53, p21ras), colon carcinoma (p21ras,HER2/neu, c-erbB-2, MUC family), colorectal cancer (Colorectalassociated antigen (CRC)-CO17-1A/GA733, APC), choriocarcinoma (CEA),epithelial cell cancer (cyclophilin b), gastric cancer (HER2/neu,c-erbB-2, ga733 glycoprotein), hepatocellular cancer (α-fetoprotein),Hodgkins lymphoma (Imp-1, EBNA-1), lung cancer (CEA, MAGE-3, NY-ESO-1),lymphoid cell-derived leukemia (cyclophilin b), melanoma (p5 protein,gp75, oncofetal antigen, GM2 and GD2 gangliosides, Melan-A/MART-1,cdc27, MAGE-3, p21ras, gp100), mycloma (MUC family, p21ras), non-smallcell lung carcinoma (HER2/neu, c-erbB-2), nasopharyngeal cancer (Imp-1,EBNA-1), ovarian cancer (MUC family, HER2/neu, c-erbB-2), prostatecancer (Prostate Specific Antigen (PSA) and its antigenic epitopesPSA-1, PSA-2, and PSA-3, PSMA, HER2/neu, c-erbB-2, ga733 glycoprotein),renal cancer (HER2/neu, c-erbB-2), squamous cell cancers of the cervixand esophagus, testicular cancer (NY-ESO-1), and T cell leukemia (HTLV-1epitopes), and viral products or proteins.

In some embodiments, the immune effector cell comprising a CAR molecule(e.g., CAR T cell) useful in the methods disclosed herein expresses aCAR comprising a mesothelin binding domain (i.e., the CAR T cellspecifically recognizes mesothelin). Mesothelin is a tumor antigen thatis overexpressed in a variety of cancers including ovarian, lung andpancreatic cancers.

In some embodiments, the immune effector cell comprising a CAR molecule(e.g., CAR T cell) useful in the methods disclosed herein expresses aCAR comprising a CD19 binding domain. In some embodiments, the immuneeffector cell comprising a CAR molecule (e.g., CAR T cell) useful in themethods disclosed herein expresses a CAR comprising a HER2 bindingdomain. In some embodiments, the immune effector cell comprising a CARmolecule (e.g., CAR T cell) useful in the methods disclosed hereinexpresses a CAR comprising an EGFR binding domain.

In some embodiments, the CAR effector cell expressing a CAR comprising aCD19 targeting or binding domain is Kymriah™ (tisagenlecleucel;Novartis; see WO 2016109410, herein incorporated by reference in itsentirety) or Yescarta™ (axicabtagene ciloleucel; Kite; see US20160346326, herein incorporated by reference in its entirety).

Linker

Provided herein are CARs that can optionally include a linker (1)between the antigen binding domain and the transmembrane domain, and/or(2) between the transmembrane domain and the cytoplasmic signalingdomain. In some embodiments, the linker can be a polypeptide linker. Forexample, the linker can have a length of between about 1 amino acid andabout 500 amino acids, about 400 amino acids, about 300 amino acids,about 200 amino acids, about 100 amino acids, about 90 amino acids,about 80 amino acids, about 70 amino acids, about 60 amino acids, about50 amino acids, about 40 amino acids, about 35 amino acids, about 30amino acids, about 25 amino acids, about 20 amino acids, about 18 aminoacids, about 16 amino acids, about 14 amino acids, about 12 amino acids,about 10 amino acids, about 8 amino acids, about 6 amino acids, about 4amino acids, or about 2 amino acids; about 2 amino acids to about 500amino acids, about 400 amino acids, about 300 amino acids, about 200amino acids, about 100 amino acids, about 90 amino acids, about 80 aminoacids, about 70 amino acids, about 60 amino acids, about 50 amino acids,about 40 amino acids, about 35 amino acids, about 30 amino acids, about25 amino acids, about 20 amino acids, about 18 amino acids, about 16amino acids, about 14 amino acids, about 12 amino acids, about 10 aminoacids, about 8 amino acids, about 6 amino acids, or about 4 amino acids;about 4 amino acids to about 500 amino acids, about 400 amino acids,about 300 amino acids, about 200 amino acids, about 100 amino acids,about 90 amino acids, about 80 amino acids, about 70 amino acids, about60 amino acids, about 50 amino acids, about 40 amino acids, about 35amino acids, about 30 amino acids, about 25 amino acids, about 20 aminoacids, about 18 amino acids, about 16 amino acids, about 14 amino acids,about 12 amino acids, about 10 amino acids, about 8 amino acids, orabout 6 amino acids; about 6 amino acids to about 500 amino acids, about400 amino acids, about 300 amino acids, about 200 amino acids, about 100amino acids, about 90 amino acids, about 80 amino acids, about 70 aminoacids, about 60 amino acids, about 50 amino acids, about 40 amino acids,about 35 amino acids, about 30 amino acids, about 25 amino acids, about20 amino acids, about 18 amino acids, about 16 amino acids, about 14amino acids, about 12 amino acids, about 10 amino acids, or about 8amino acids; about 8 amino acids to about 500 amino acids, about 400amino acids, about 300 amino acids, about 200 amino acids, about 100amino acids, about 90 amino acids, about 80 amino acids, about 70 aminoacids, about 60 amino acids, about 50 amino acids, about 40 amino acids,about 35 amino acids, about 30 amino acids, about 25 amino acids, about20 amino acids, about 18 amino acids, about 16 amino acids, about 14amino acids, about 12 amino acids, or about 10 amino acids; about 10amino acids to about 500 amino acids, about 400 amino acids, about 300amino acids, about 200 amino acids, about 100 amino acids, about 90amino acids, about 80 amino acids, about 70 amino acids, about 60 aminoacids, about 50 amino acids, about 40 amino acids, about 35 amino acids,about 30 amino acids, about 25 amino acids, about 20 amino acids, about18 amino acids, about 16 amino acids, about 14 amino acids, or about 12amino acids; about 12 amino acids to about 500 amino acids, about 400amino acids, about 300 amino acids, about 200 amino acids, about 100amino acids, about 90 amino acids, about 80 amino acids, about 70 aminoacids, about 60 amino acids, about 50 amino acids, about 40 amino acids,about 35 amino acids, about 30 amino acids, about 25 amino acids, about20 amino acids, about 18 amino acids, about 16 amino acids, or about 14amino acids; about 14 amino acids to about 500 amino acids, about 400amino acids, about 300 amino acids, about 200 amino acids, about 100amino acids, about 90 amino acids, about 80 amino acids, about 70 aminoacids, about 60 amino acids, about 50 amino acids, about 40 amino acids,about 35 amino acids, about 30 amino acids, about 25 amino acids, about20 amino acids, about 18 amino acids, or about 16 amino acids; about 16amino acids to about 500 amino acids, about 400 amino acids, about 300amino acids, about 200 amino acids, about 100 amino acids, about 90amino acids, about 80 amino acids, about 70 amino acids, about 60 aminoacids, about 50 amino acids, about 40 amino acids, about 35 amino acids,about 30 amino acids, about 25 amino acids, about 20 amino acids, orabout 18 amino acids; about 18 amino acids to about 500 amino acids,about 400 amino acids, about 300 amino acids, about 200 amino acids,about 100 amino acids, about 90 amino acids, about 80 amino acids, about70 amino acids, about 60 amino acids, about 50 amino acids, about 40amino acids, about 35 amino acids, about 30 amino acids, about 25 aminoacids, or about 20 amino acids; about 20 amino acids to about 500 aminoacids, about 400 amino acids, about 300 amino acids, about 200 aminoacids, about 100 amino acids, about 90 amino acids, about 80 aminoacids, about 70 amino acids, about 60 amino acids, about 50 amino acids,about 40 amino acids, about 35 amino acids, about 30 amino acids, orabout 25 amino acids; about 25 amino acids to about 500 amino acids,about 400 amino acids, about 300 amino acids, about 200 amino acids,about 100 amino acids, about 90 amino acids, about 80 amino acids, about70 amino acids, about 60 amino acids, about 50 amino acids, about 40amino acids, about 35 amino acids, or about 30 amino acids; about 30amino acids to about 500 amino acids, about 400 amino acids, about 300amino acids, about 200 amino acids, about 100 amino acids, about 90amino acids, about 80 amino acids, about 70 amino acids, about 60 aminoacids, about 50 amino acids, about 40 amino acids, or about 35 aminoacids; about 35 amino acids to about 500 amino acids, about 400 aminoacids, about 300 amino acids, about 200 amino acids, about 100 aminoacids, about 90 amino acids, about 80 amino acids, about 70 amino acids,about 60 amino acids, about 50 amino acids, or about 40 amino acids;about 40 amino acids to about 500 amino acids, about 400 amino acids,about 300 amino acids, about 200 amino acids, about 100 amino acids,about 90 amino acids, about 80 amino acids, about 70 amino acids, about60 amino acids, or about 50 amino acids; about 50 amino acids to about500 amino acids, about 400 amino acids, about 300 amino acids, about 200amino acids, about 100 amino acids, about 90 amino acids, about 80 aminoacids, about 70 amino acids, or about 60 amino acids; about 60 aminoacids to about 500 amino acids, about 400 amino acids, about 300 aminoacids, about 200 amino acids, about 150 amino acids, about 100 aminoacids, about 90 amino acids, about 80 amino acids, or about 70 aminoacids; about 70 amino acids to about 500 amino acids, about 400 aminoacids, about 300 amino acids, about 200 amino acids, about 100 aminoacids, about 90 amino acids, or about 80 amino acids; about 80 aminoacids to about 500 amino acids, about 400 amino acids, about 300 aminoacids, about 200 amino acids, about 100 amino acids, or about 90 aminoacids; about 90 amino acids to about 500 amino acids, about 400 aminoacids, about 300 amino acids, about 200 amino acids, or about 100 aminoacids; about 100 amino acids to about 500 amino acids, about 400 aminoacids, about 300 amino acids, or about 200 amino acids; about 200 aminoacids to about 500 amino acids, about 400 amino acids, or about 300amino acids; about 300 amino acids to about 500 amino acids or about 400amino acids; or about 400 amino acids to about 500 amino acids.

Additional examples and aspects of linkers are described in thereferences cited herein, and are thus incorporated in their entiretyherein.

Transmembrane Domains

In some embodiments, the CARs described herein also include atransmembrane domain. In some embodiments, the transmembrane domain isnaturally associated with a sequence in the cytoplasmic domain. In someembodiments, the transmembrane domain can be modified by one or more(e.g., two, three, four, five, six, seven, eight, nine, or ten) aminoacid substitutions to avoid the binding of the domain to othertransmembrane domains (e.g., the transmembrane domains of the same ordifferent surface membrane proteins) to minimize interactions with othermembers of the receptor complex.

In some embodiments, the transmembrane domain may be derived from anatural source. In some embodiments, the transmembrane domain may bederived from any membrane-bound or transmembrane protein. Non-limitingexamples of transmembrane domains that may be used herein may be derivedfrom (e.g., comprise at least the transmembrane sequence or a part ofthe transmembrane sequence of) the alpha, beta, or zeta chain of theT-cell receptor, CD28, CD3 epsilon, CD33, CD37, CD64, CD80, CD45, CD4,CD5, CD8, CD9, CD16, CD22, CD86, CD134, CD137 or CD154.

In some embodiments, the transmembrane domain may be synthetic. Forexample, in some embodiments where the transmembrane domain is from asynthetic source, the transmembrane domain may include (e.g.,predominantly include) hydrophobic residues (e.g., leucine and valine).In some embodiments, the synthetic transmembrane domain will include atleast one (e.g., at least two, at least three, at least four, at leastfive, or at least six) triplet of phenylalanine, tryptophan, and valineat the end of a synthetic transmembrane domain. In some embodiments, thetransmembrane domain of a CAR can include a CD8 hinge domain.

Additional specific examples of transmembrane domains are described inthe references cited herein.

Cytoplasmic Domains

Also provided herein are CAR molecules that comprise, e.g., acytoplasmic signaling domain that includes a cytoplasmic sequence ofCD3ζ sufficient to stimulate a T cell when the antigen binding domainbinds to the antigen, and optionally, a cytoplasmic sequence of one ormore of co-stimulatory proteins (e.g., a cytoplasmic sequence of one ormore of CD27, CD28, 4-1BB, OX40, CD30, CD40L, CD40, PD-1, PD-L1, ICOS,LFA-1, CD2, CD7, CD160, LIGHT, BTLA, TIM3, CD244, CD80, LAG3, NKG2C,B7-H3, a ligand that specifically binds to CD83, and any of the ITAMsequences described herein or known in the art) that provides forco-stimulation of the T cell. The stimulation of a CAR immune effectorcell can result in the activation of one or more anti-cancer activitiesof the CAR immune effector cell. For example, in some embodiments,stimulation of a CAR immune effector cell can result in an increase inthe cytolytic activity or helper activity of the CAR immune effectorcell, including the secretion of cytokines. In some embodiments, theentire intracellular signaling domain of a co-stimulatory protein isincluded in the cytoplasmic signaling domain. In some embodiments, thecytoplasmic signaling domain includes a truncated portion of anintracellular signaling domain of a co-stimulatory protein (e.g., atruncated portion of the intracellular signaling domain that transducesan effector function signal in the CAR immune effector cell).Non-limiting examples of intracellular signaling domains that can beincluded in a cytoplasmic signaling domain include the cytoplasmicsequences of the T cell receptor (TCR) and co-receptors that act inconcert to initiate signal transduction following antigen receptorengagement, as well as any variant of these sequences including at leastone (e.g., one, two, three, four, five, six, seven, eight, nine, or ten)substitution and have the same or about the same functional capability.

In some embodiments, a cytoplasmic signaling domain can include twodistinct classes of cytoplasmic signaling sequences: signaling sequencesthat initiate antigen-dependent activation through the TCR (primarycytoplasmic signaling sequences) (e.g., a CD3ζ cytoplasmic signalingsequence) and a cytoplasmic sequence of one or more of co-stimulatoryproteins that act in an antigen-independent manner to provide asecondary or co-stimulatory signal (secondary cytoplasmic signalingsequences).

In some embodiments, the cytoplasmic domain of a CAR can be designed toinclude the CD3ζ signaling domain by itself or combined with any otherdesired cytoplasmic signaling sequence(s) useful in the context of aCAR. In some examples, the cytoplasmic domain of a CAR can include aCD3ζ chain portion and a costimulatory cytoplasmic signaling sequence.

The costimulatory cytoplasmic signaling sequence refers to a portion ofa CAR including a cytoplasmic signaling sequence of a costimulatoryprotein (e.g., CD27, CD28, 4-IBB (CD 137), OX40, CD30, CD40, PD-1, ICOS,lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT,NKG2C, B7-H3, and a ligand that specifically binds with CD83).

In some embodiments, the cytoplasmic signaling sequences within thecytoplasmic signaling domain of a CAR are positioned in a random order.In some embodiments, the cytoplasmic signaling sequences within thecytoplasmic signaling domain of a CAR are linked to each other in aspecific order. In some embodiments, a linker (e.g., any of the linkersdescribed herein) can be used to form a linkage between differentcytoplasmic signaling sequences.

In some embodiments, the cytoplasmic signaling domain is designed toinclude the cytoplasmic signaling sequence of CD3ζ and the cytoplasmicsignaling sequence of the costimulatory protein CD28. In someembodiments, the cytoplasmic signaling domain is designed to include thecytoplasmic signaling sequence of CD3ζ and the cytoplasmic signalingsequence of costimulatory protein 4-IBB. In some embodiments, thecytoplasmic signaling domain is designed to include the cytoplasmicsignaling sequence of CD3ζ and the cytoplasmic signaling sequences ofcostimulatory proteins CD28 and 4-1BB. In some embodiments, thecytoplasmic signaling domain does not include the cytoplasmic signalingsequences of 4-1BB.

Additional Modification of CAR T Cells

In another embodiment, the therapeutic efficacy of CAR effector cells(e.g., CAR T cells) is enhanced by disruption of a methylcytosinedioxygenase gene (e.g., Tet1, Tet2, Tet3), which leads to decreasedtotal levels of 5-hydroxymethylcytosine in association with enhancedproliferation, regulation of effector cytokine production anddegranulation, and thereby increases CAR effector cell (e.g., CAR Tcell) proliferation and/or function, as described in PCT Publication WO2017/049166. Thus, an effector cell (e.g., T cell) can be engineered toexpress a CAR and wherein expression and/or function of Tet1, Tet2and/or Tet3 in said effector cell (e.g., T cell) has been reduced oreliminated.

In another embodiment, the therapeutic efficacy of CAR effector cells(e.g., CAR T cells) is enhanced by using an effector cell (e.g., T cell)that constitutively expresses a CAR (referred to as a nonconditionalCAR) and conditionally expresses another agent useful for treatingcancer, as described in PCT Publication WO 2016/126608 and USPublication No. 2018/0044424. In such embodiments, the conditionallyexpressed agent is expressed upon activation of the effector cell (e.g.,T cell), e.g., the binding of the nonconditional CAR to its target. Inone embodiment, the conditionally expressed agent is a CAR (referred toherein as a conditional CAR). In another embodiment, the conditionallyexpressed agent inhibits a checkpoint inhibitor of the immune response.In another embodiment, the conditionally expressed agent improves orenhances the efficacy of a CAR, and can include a cytokine.

In another embodiment, the therapeutic efficacy of CAR T cells isenhanced by modifying the CAR T cell with a nucleic acid that is capableof altering (e.g., downmodulating) expression of an endogenous geneselected from the group consisting of TCR a chain, TCR 3 chain, beta-2microglobulin, a HLA molecule, CTLA-4, PDI, and FAS, as described in PCTPublication WO 2016/069282 and US Publication No. 2017/0335331.

In another embodiment, the therapeutic efficacy of CAR T cells isenhanced by co-expressing in the T cells the CAR and one or moreenhancers of T cell priming (“ETPs”), as described in PCT Publication WO2015/112626 and US Publication No. 2016/0340406. The addition of an ETPcomponent to the CAR T cell confers enhanced “professional”antigen-presenting cell (APC) function. In an embodiment, the CAR andone or more ETPs are transiently co-expressed in the T cell. Thus, theengineered T cells are safe (given the transient nature of the CARIETPexpression), and induce prolonged immunity via APC function.

In another embodiment, the therapeutic efficacy of CAR T cells isenhanced by co-expressing in the T cells a CAR and an inhibitorymembrane protein (IMP) comprising a binding (or dimerization) domain, asdescribed in PCT Publication WO 2016/055551 and US Publication No.2017/0292118. The CAR and the IMP are made both reactive to a solublecompound, especially through a second binding domain comprised withinthe CAR, thereby allowing the co-localization, by dimerization or ligandrecognition, of the inhibitory signaling domain borne by the IMP and ofthe signal transducing domain borne by the CAR, having the effect ofturning down the CAR activation. The inhibitory signaling domain ispreferably the programmed death-1 (PD-1), which attenuates T-cellreceptor (TCR)-mediated activation of IL-2 production and T-cellproliferation.

In another embodiment, the therapeutic efficacy of CAR T cells isenhanced using a system where controlled variations in the conformationof the extracellular portion of a CAR containing the antigen-bindingdomain is obtained upon addition of small molecules, as described in PCTPublication WO 2017/032777. This integrated system switches theinteraction between the antigen and the antigen binding domain betweenon/off states. By being able to control the conformation of theextracellular portion of a CAR, downstream functions of the CAR T cell,such as cytotoxicity, can be directly modulated. Thus, a CAR can becharacterized in that it comprises: a) at least one ectodomain whichcomprises: i) an extracellular antigen binding domain; and ii) a switchdomain comprising at least a first multimerizing ligand-binding domainand a second multimerizing ligand-binding domain which are capable ofbinding to a predetermined multivalent ligand to form a multimercomprising said two binding domains and the multivalent ligand to whichthey are capable of binding; b) at least one transmembrane domain; andc) at least one endodomain comprising a signal transducing domain andoptionally a co-stimulatory domain; wherein the switch domain is locatedbetween the extracellular antigen binding domain and the transmembranedomain.

II. Tumor-Associated Antigen Targeting Antibodies

In some aspects, the disclosure provides immunomodulatory fusionproteins to be used or performed in conjunction with antibodies thattarget tumor antigens.

Therapeutic monoclonal antibodies have been conceived as a class ofpharmaceutically active agents which should allow tumor selectivetreatment by targeting tumor selective antigens or epitopes.

Methods of producing antibodies, and antigen-binding fragments thereof,are well known in the art and are disclosed in, e.g., U.S. Pat. Nos.7,247,301, 7,923,221, and U.S. Patent Application 2008/0138336, all ofwhich are herein incorporated by reference in their entirety.

Therapeutic antibodies that can be used in the methods of the presentdisclosure include, but are not limited to, any of the art-recognizedanti-cancer antibodies that are approved for use, in clinical trials, orin development for clinical use. In certain embodiments, more than oneanti-cancer antibody can be included in the combination therapy of thepresent disclosure.

Non-limiting examples of anti-cancer antibodies include the following,without limitation: trastuzumab (HERCEPTIN™, by Genentech, South SanFrancisco, Calif.), which is used to treat HER-2/neu positive breastcancer or metastatic breast cancer; bevacizumab (AVASTIN™ by Genentech),which are used to treat colorectal cancer, metastatic colorectal cancer,breast cancer, metastatic breast cancer, non-small cell lung cancer, orrenal cell carcinoma; rituximab (RITUXAN™ by Genentech), which is usedto treat non-Hodgkin's lymphoma or chronic lymphocytic leukemia;pertuzumab (OMNITARG™ by Genentech), which is used to treat breastcancer, prostate cancer, non-small cell lung cancer, or ovarian cancer;cetuximab (ERBITUX™ by ImClone Systems Incorporated, New York, N.Y.),which can be used to treat colorectal cancer, metastatic colorectalcancer, lung cancer, head and neck cancer, colon cancer, breast cancer,prostate cancer, gastric cancer, ovarian cancer, brain cancer,pancreatic cancer, esophageal cancer, renal cell cancer, prostatecancer, cervical cancer, or bladder cancer; IMC-1C11 (ImClone SystemsIncorporated), which is used to treat colorectal cancer, head and neckcancer, as well as other potential cancer targets; tositumomab andtositumomab and iodine I 131 (BEXXAR XM by Corixa Corporation, Seattle,Wash.), which is used to treat non-Hodgkin's lymphoma, which can be CD20positive, follicular, non-Hodgkin's lymphoma, with and withouttransformation, whose disease is refractory to Rituximab and hasrelapsed following chemotherapy; In¹¹¹ ibirtumomab tiuxetan; Y⁹⁰ibirtumomab tiuxetan; In¹¹¹ ibirtumomab tiuxetan and Y⁹⁰ ibirtumomabtiuxetan (ZEVALIN™ by Biogen Idee, Cambridge, Mass.), which is used totreat lymphoma or non-Hodgkin's lymphoma, which can include relapsedfollicular lymphoma; relapsed or refractory, low grade or follicularnon-Hodgkin's lymphoma; or transformed B-cell non-Hodgkin's lymphoma;EMD 7200 (EMD Pharmaceuticals, Durham, N.C.), which is used for treatingnon-small cell lung cancer or cervical cancer; SGN-30 (a geneticallyengineered monoclonal antibody targeted to CD30 antigen by SeattleGenetics, Bothell, Wash.), which is used for treating Hodgkin's lymphomaor non-Hodgkin's lymphoma; SGN-15 (a genetically engineered monoclonalantibody targeted to a Lewisy-related antigen that is conjugated todoxorubicin by Seattle Genetics), which is used for treating non-smallcell lung cancer; SGN-33 (a humanized antibody targeted to CD33 antigenby Seattle Genetics), which is used for treating acute myeloid leukemia(AML) and myelodysplasia syndromes (MDS); SGN-40 (a humanized monoclonalantibody targeted to CD40 antigen by Seattle Genetics), which is usedfor treating multiple myeloma or non-Hodgkin's lymphoma;

SGN-35 (a genetically engineered monoclonal antibody targeted to a CD30antigen that is conjugated to auristatin E by Seattle Genetics), whichis used for treating non-Hodgkin's lymphoma; SGN-70 (a humanizedantibody targeted to CD70 antigen by Seattle Genetics), which is usedfor treating renal cancer and nasopharyngeal carcinoma; SGN-75 (aconjugate comprised of the SGN70 antibody and an Auristatin derivativeby Seattle Genetics); and SGN-17/19 (a fusion protein containingantibody and enzyme conjugated to melphalan prodrug by SeattleGenetics), which is used for treating melanoma or metastatic melanoma.

It should be understood that the therapeutic antibodies to be used inthe methods of the present disclosure are not limited to those describedsupra. For example, the following approved therapeutic antibodies canalso be used in the methods of the disclosure: brentuximab vedotin(ADCETRIS™) for anaplastic large cell lymphoma and Hodgkin lymphoma,ipilimumab (MDX-101; YERVOY™) for melanoma, ofatumumab (ARZERRA™) forchronic lymphocytic leukemia, panitumumab (VECTIBIX™) for colorectalcancer, alemtuzumab (CAMPATH™) for chronic lymphocytic leukemia,ofatumumab (ARZERRA™) for chronic lymphocytic leukemia, gemtuzumabozogamicin (MYLOTARG™) for acute myelogenous leukemia.

Antibodies suitable for use in the methods disclosed herein can alsotarget molecules expressed by immune cells, such as, but not limited to,0X86 which targets OX40 and increases antigen-specific CD8+ T cells attumor sites and enhances tumor rejection; BMS-663513 which targets CD137and causes regression of established tumors, as well as the expansionand maintenance of CD8+ T cells, and daclizumab (ZENAPAX™) which targetsCD25 and causes transient depletion of CD4+CD25+FOXP3+Tregs and enhancestumor regression and increases the number of effector T cells. A moredetailed discussion of these antibodies can be found in, e.g., Weiner etal., Nature Rev. Immunol 2010; 10:317-27.

Other therapeutic antibodies can be identified that target tumorantigens (e.g., tumor antigens associated with different types ofcancers, such as carcinomas, sarcomas, myelomas, leukemias, lymphomas,and combinations thereof). For example, the following tumor antigens canbe targeted by therapeutic antibodies in the methods disclosed herein.

The tumor antigen may be an epithelial cancer antigen, (e.g., breast,gastrointestinal, lung), a prostate specific cancer antigen (PSA) orprostate specific membrane antigen (PSMA), a bladder cancer antigen, alung (e.g., small cell lung) cancer antigen, a colon cancer antigen, anovarian cancer antigen, a brain cancer antigen, a gastric cancerantigen, a renal cell carcinoma antigen, a pancreatic cancer antigen, aliver cancer antigen, an esophageal cancer antigen, a head and neckcancer antigen, or a colorectal cancer antigen. In certain embodiments,the tumor antigen is a lymphoma antigen (e.g., non-Hodgkin's lymphoma orHodgkin's lymphoma), a B-cell lymphoma cancer antigen, a leukemiaantigen, a myeloma (e.g., multiple myeloma or plasma cell myeloma)antigen, an acute lymphoblastic leukemia antigen, a chronic myeloidleukemia antigen, or an acute myelogenous leukemia antigen. It should beunderstood that the described tumor antigens are only exemplary and thatany tumor antigen can be targeted for use in the methods disclosedherein.

In certain embodiments, the tumor antigen is a mucin-1 protein orpeptide (MUC-1) that is found on most or all human adenocarcinomas:pancreas, colon, breast, ovarian, lung, prostate, head and neck,including multiple myelomas and some B cell lymphomas. Patients withinflammatory bowel disease, either Crohn's disease or ulcerativecolitis, are at an increased risk for developing colorectal carcinoma.MUC-1 is a type I transmembrane glycoprotein. The major extracellularportion of MUC-1 has a large number of tandem repeats consisting of 20amino acids which comprise immunogenic epitopes. In some cancers it isexposed in an unglycosylated form that is recognized by the immunesystem (Gendler et al., J Biol Chem 1990; 265:15286-15293).

In certain embodiments, the tumor antigen is a mutated B-Raf antigen,which is associated with melanoma and colon cancer. The vast majority ofthese mutations represent a single nucleotide change of T-A atnucleotide 1796 resulting in a valine to glutamic acid change at residue599 within the activation segment of B-Raf. Raf proteins are alsoindirectly associated with cancer as effectors of activated Rasproteins, oncogenic forms of which are present in approximatelyone-third of all human cancers. Normal non-mutated B-Raf is involved incell signaling, relaying signals from the cell membrane to the nucleus.The protein is usually only active when needed to relay signals. Incontrast, mutant B-Raf has been reported to be constantly active,disrupting the signaling relay (Mercer and Pritchard, Biochim BiophysActa (2003) 1653(1):25-40; Sharkey et al., Cancer Res. (2004)64(5):1595-1599).

In certain embodiments, the tumor antigen is a human epidermal growthfactor receptor-2 (HER-2/neu) antigen. Cancers that have cells thatoverexpress HER-2/neu are referred to as HER-2/neu⁺ cancers. ExemplaryHER-2/neu⁺ cancers include prostate cancer, lung cancer, breast cancer,ovarian cancer, pancreatic cancer, skin cancer, liver cancer (e.g.,hepatocellular adenocarcinoma), intestinal cancer, and bladder cancer.

HER-2/neu has an extracellular binding domain (ECD) of approximately 645aa, with 40% homology to epidermal growth factor receptor (EGFR), ahighly hydrophobic transmembrane anchor domain (TMD), and acarboxyterminal intracellular domain (ICD) of approximately 580 aa with80% homology to EGFR. The nucleotide sequence of HER-2/neu is availableat GENBANK™. Accession Nos. AH002823 (human HER-2 gene, promoter regionand exon 1); M16792 (human HER-2 gene, exon 4): M16791 (human HER-2gene, exon 3); M16790 (human HER-2 gene, exon 2); and M16789 (humanHER-2 gene, promoter region and exon 1). The amino acid sequence for theHER-2/neu protein is available at GENBANK™ Accession No. AAA58637. Basedon these sequences, one skilled in the art could develop HER-2/neuantigens using known assays to find appropriate epitopes that generatean effective immune response. Exemplary HER-2/neu antigens includep369-377 (a HER-2/neu derived HLA-A2 peptide); dHER2 (CorixaCorporation); li-Key MHC class II epitope hybrid (Generex BiotechnologyCorporation); peptide P4 (amino acids 378-398); peptide P7 (amino acids610-623); mixture of peptides P6 (amino acids 544-560) and P7; mixtureof peptides P4, P6 and P7; HER2 [9₇₅₄]; and the like.

In certain embodiments, the tumor antigen is an epidermal growth factorreceptor (EGFR) antigen. The EGFR antigen can be an EGFR variant 1antigen, an EGFR variant 2 antigen, an EGFR variant 3 antigen and/or anEGFR variant 4 antigen. Cancers with cells that overexpress EGFR arereferred to as EGFR⁺ cancers. Exemplary EGFR⁺ cancers include lungcancer, head and neck cancer, colon cancer, colorectal cancer, breastcancer, prostate cancer, gastric cancer, ovarian cancer, brain cancerand bladder cancer.

In certain embodiments, the tumor antigen is a vascular endothelialgrowth factor receptor (VEGFR) antigen. VEGFR is considered to be aregulator of cancer-induced angiogenesis. Cancers with cells thatoverexpress VEGFR are called VEGFR⁺ cancers. Exemplary VEGFR⁺ cancersinclude breast cancer, lung cancer, small cell lung cancer, coloncancer, colorectal cancer, renal cancer, leukemia, and lymphocyticleukemia.

In certain embodiments, the tumor antigen is prostate-specific antigen(PSA) and/or prostate-specific membrane antigen (PSMA) that areprevalently expressed in androgen-independent prostate cancers.

In certain embodiments, the tumor antigen is Glycoprotein 100 (gp 100),a tumor-specific antigen associated with melanoma.

In certain embodiments, the tumor antigen is a carcinoembryonic (CEA)antigen. Cancers with cells that overexpress CEA are referred to as CEA⁺cancers. Exemplary CEA⁺ cancers include colorectal cancer, gastriccancer and pancreatic cancer. Exemplary CEA antigens include CAP-1(i.e., CEA aa 571-579), CAP1-6D, CAP-2 (i.e., CEA aa 555-579), CAP-3(i.e., CEA aa 87-89), CAP-4 (CEA aa 1-11), CAP-5 (i.e., CEA aa 345-354),CAP-6 (i.e., CEA aa 19-28) and CAP-7.

In certain embodiments, the tumor antigen is carbohydrate antigen 10.9(CA 19.9). CA 19.9 is an oligosaccharide related to the Lewis A bloodgroup substance and is associated with colorectal cancers.

In certain embodiments, the tumor antigen is a melanoma cancer antigen.Melanoma cancer antigens are useful for treating melanoma. Exemplarymelanoma cancer antigens include MART-1 (e.g., MART-1 26-35 peptide,MART-1 27-35 peptide); MART-1/Melan A; pMel17; pMel17/gp100; gp100(e.g., gp 100 peptide 280-288, gp 100 peptide 154-162, gp 100 peptide457-467); TRP-1; TRP-2; NY-ESO-1; p16; beta-catenin; mum-1; and thelike.

In certain embodiments, the tumor antigen is a mutant or wild type raspeptide. The mutant ras peptide can be a mutant K-ras peptide, a mutantN-ras peptide and/or a mutant H-ras peptide. Mutations in the rasprotein typically occur at positions 12 (e.g., arginine or valinesubstituted for glycine), 13 (e.g., asparagine for glycine), 61 (e.g.,glutamine to leucine) and/or 59. Mutant ras peptides can be useful aslung cancer antigens, gastrointestinal cancer antigens, hepatomaantigens, myeloid cancer antigens (e.g., acute leukemia,myelodysplasia), skin cancer antigens (e.g., melanoma, basal cell,squamous cell), bladder cancer antigens, colon cancer antigens,colorectal cancer antigens, and renal cell cancer antigens.

In certain embodiments, the tumor antigen is a mutant and/or wildtypep53 peptide. The p53 peptide can be used as colon cancer antigens, lungcancer antigens, breast cancer antigens, hepatocellular carcinoma cancerantigens, lymphoma cancer antigens, prostate cancer antigens, thyroidcancer antigens, bladder cancer antigens, pancreatic cancer antigens andovarian cancer antigens.

Further tumor antigens are well known in the art and include, forexample, a glioma-associated antigen, carcinoembryonic antigen (CEA),β-human chorionic gonadotropin, alphafetoprotein (AFP), lectin-reactiveAFP, thyroglobulm, RAGE-1, MN-CA IX, human telomerase reversetranscriptase, RU1, RU2 (AS), intestinal carboxy esterase, mut hsp70-2,M-CSF, prostase, prostate-specific antigen (PSA), PAP, NY-ESO-1,LAGE-1a, p53, tyrosinase, prostein, PSMA, ras, Her2/neu, TRP-1, TRP-2,TAG-72, KSA, CA-125, PSA, BRCI, BRC-II, bcr-abl, pax3-fkhr, ews-fli-1,survivin and telomerase, prostate-carcinoma tumor antigen-1 (PCTA-1),MAGE, GAGE, GP-100, MUC-1, MUC-2, ELF2M, neutrophil elastase, ephrinB2,CD22, insulin growth factor (IGF)-I, IGF-II, IGF-I receptor, andmesothelin,

In certain embodiments, the tumor antigen comprises one or moreantigenic cancer epitopes associated with a malignant tumor. Malignanttumors express a number of proteins that can serve as target antigensfor an immune attack. These molecules include but are not limited totissue-specific antigens such as MART-1, tyrosinase and GP 100 inmelanoma and prostatic acid phosphatase (PAP) and prostate-specificantigen (PSA) in prostate cancer. Other target molecules belong to thegroup of transformation-related molecules such as the oncogene HER-2/NeuErbB-2. Yet another group of target antigens are onco-fetal antigenssuch as carcinoembryonic antigen (CEA). In B-cell lymphoma thetumor-specific idiotype immunoglobulin constitutes a trulytumor-specific immunoglobulin antigen that is unique to the individualtumor. B-cell differentiation antigens such as CD19, CD20 and CD37 areother candidates for target antigens in B-cell lymphoma. Some of theseantigens (CEA, HER-2, CD19, CD20, idiotype) have been used as targetsfor passive immunotherapy with monoclonal antibodies with limitedsuccess.

The tumor antigen may also be a tumor-specific antigen (TSA) or atumor-associated antigen (TAA). A TSA is unique to tumor cells and doesnot occur on other cells in the body. A TAA associated antigen is notunique to a tumor cell and instead is also expressed on a normal cellunder conditions that fail to induce a state of immunologic tolerance tothe antigen. The expression of the antigen on the tumor may occur underconditions that enable the immune system to respond to the antigen. TAAsmay be antigens that are expressed on normal cells during fetaldevelopment when the immune system is immature and unable to respond orthey may be antigens that are normally present at extremely low levelson normal cells but which are expressed at much higher levels on tumorcells.

Non-limiting examples of TSA or TAA antigens include the following:Differentiation antigens such as MART-1/MelanA (MART-1), Pmel 17,tyrosinase, TRP-1, TRP-2 and tumor-specific multilineage antigens suchas MAGE-1, MAGE-3, BAGE, GAGE-1, GAGE-2, pi 5; overexpressed embryonicantigens such as CEA; overexpressed oncogenes and mutatedtumor-suppressor genes such as p53, Ras, HER-2/neu; unique tumorantigens resulting from chromosomal translocations such as BCR-ABL,E2A-PRL, H4-RET, 1GH-IGK, MYL-RAR; and viral antigens, such as theEpstein Barr virus antigens EBVA and the human papillomavirus (HPV)antigens E6 and E7. Other large, protein-based antigens include TSP-180,MAGE-4, MAGE-5, MAGE-6, RAGE, NY-ESO, p185erbB2, p180erbB-3, c-met,nm-23H1, PSA, TAG-72, CA 19-9, CA 72-4, CAM 17.1, NuMa, K-ras,beta-Catenin, CDK4, Mum-1, p 15, p 16, 43-9F, 5T4(791Tgp72}alpha-fetoprotem, beta-HCG, BCA225, BTAA, CA 125, CA 15-3\CA 27.29\BCAA,CA 195, CA 242, CA-50, CAM43, CD68\I, CO-029, FGF-5, G250, Ga733VEpCAM,HTgp-175, M344, MA-50, MG7-Ag, MOV 18, NB/70K, NY-CO-1, RCAS 1,SDCCAG16, TA-90\Mac-2 binding protein, Acyclophilin C-associatedprotein, TAAL6, TAG72, TLP, and TPS.

In certain embodiments, the tumor-associated antigen is determined bysequencing a patient's tumor cells and identifying mutated proteins onlyfound in the tumor. These antigens are referred to as “neoantigens.”Once a neoantigen has been identified, therapeutic antibodies can beproduced against it and used in the methods described herein.

The therapeutic antibody can be a fragment of an antibody; a complexcomprising an antibody; or a conjugate comprising an antibody. Theantibody can optionally be chimeric or humanized or fully human.

III. Immune Checkpoint Blockade

In some aspects, the disclosure provides immunomodulatory fusionproteins to be used or performed in conjunction with immune checkpointinhibitors or immune checkpoint blockers.

T cell activation and effector functions are balanced by co-stimulatoryand inhibitory signals, referred to as “immune checkpoints.” Inhibitoryligands and receptors that regulate T cell effector functions areoverexpressed on tumor cells. Subsequently, agonists of co-stimulatoryreceptors or antagonists of inhibitory signals, result in theamplification of antigen-specific T cell responses. In contrast totherapeutic antibodies which target tumor cells directly, immunecheckpoint blocker enhances endogenous anti-tumor activity. In certainembodiments, the immune checkpoint blocker suitable for use in themethods disclosed herein, is an antagonist of inhibitory signals, e.g.,an antibody which targets, for example, PD-1, PD-L1, CTLA-4, LAG3,B7-H3, B7-H4, or TIM3. These ligands and receptors are reviewed inPardoll, D., Nature. 12: 252-264, 2012.

In certain embodiments, the immune checkpoint blocker is an antibody oran antigen-binding portion thereof, that disrupts or inhibits signalingfrom an inhibitory immunoregulator. In certain embodiments, the immunecheckpoint blocker is a small molecule that disrupts or inhibitssignaling from an inhibitory immunoregulator.

In certain embodiments, the inhibitory immunoregulator (immunecheckpoint blocker) is a component of the PD-1/PD-L1 signaling pathway.Accordingly, certain embodiments of the disclosure provide methods forimmunotherapy of a subject afflicted with cancer, which methods compriseadministering to the subject a therapeutically effective amount of anantibody or an antigen-binding portion thereof that disrupts theinteraction between the PD-1 receptor and its ligand, PD-L1. Antibodiesknown in the art which bind to PD-1 and disrupt the interaction betweenthe PD-1 and its ligand, PD-L1, and stimulates an anti-tumor immuneresponse, are suitable for use in the methods disclosed herein. Incertain embodiments, the antibody or antigen-binding portion thereofbinds specifically to PD-1. For example, antibodies that target PD-1 andare in clinical trials include, e.g., nivolumab (BMS-936558,Bristol-Myers Squibb) and pembrolizumab (lambrolizumab, MK03475, Merck).Other suitable antibodies for use in the methods disclosed herein areanti-PD-1 antibodies disclosed in U.S. Pat. No. 8,008,449, hereinincorporated by reference. In certain embodiments, the antibody orantigen-binding portion thereof binds specifically to PD-L1 and inhibitsits interaction with PD-1, thereby increasing immune activity.Antibodies known in the art which bind to PD-L1 and disrupt theinteraction between the PD-1 and PD-L1, and stimulates an anti-tumorimmune response, are suitable for use in the methods disclosed herein.For example, antibodies that target PD-L1 and are in clinical trials,include BMS-936559 (Bristol-Myers Squibb) and MPDL3280A (Genetech).Other suitable antibodies that target PD-L1 are disclosed in U.S. Pat.No. 7,943,743. It will be understood by one of ordinary skill that anyantibody which binds to PD-1 or PD-L1, disrupts the PD-1/PD-L1interaction, and stimulates an anti-tumor immune response, is suitablefor use in the methods disclosed herein.

In certain embodiments, the inhibitory immunoregulator is a component ofthe CTLA-4 signaling pathway. Accordingly, certain embodiments of thedisclosure provide methods for immunotherapy of a subject afflicted withcancer, which methods comprise administering to the subject atherapeutically effective amount of an antibody or an antigen-bindingportion thereof that targets CTLA-4 and disrupts its interaction withCD80 and CD86. Exemplary antibodies that target CTLA-4 includeipilimumab (MDX-010, MDX-101, Bristol-Myers Squibb), which is FDAapproved, and tremelimumab (ticilimumab, CP-675, 206, Pfizer), currentlyundergoing human trials. Other suitable antibodies that target CTLA-4are disclosed in WO 2012/120125, U.S. Pat. Nos. 6,984,720, 6,682,7368,and U.S. Patent Applications 2002/0039581, 2002/0086014, and2005/0201994, herein incorporated by reference. It will be understood byone of ordinary skill that any antibody which binds to CTLA-4, disruptsits interaction with CD80 and CD86, and stimulates an anti-tumor immuneresponse, is suitable for use in the methods disclosed herein.

In certain embodiments, the inhibitory immunoregulator is a component ofthe LAG3 (lymphocyte activation gene 3) signaling pathway. Accordingly,certain embodiments of the disclosure provide methods for immunotherapyof a subject afflicted with cancer, which methods comprise administeringto the subject a therapeutically effective amount of an antibody or anantigen-binding portion thereof that targets LAG3 and disrupts itsinteraction with MHC class II molecules. An exemplary antibody thattargets LAG3 is IMP321 (Immutep), currently undergoing human trials.Other suitable antibodies that target LAG3 are disclosed in U.S. PatentApplication 2011/0150892, herein incorporated by reference. It will beunderstood by one of ordinary skill that any antibody which binds toLAG3, disrupts its interaction with MHC class II molecules, andstimulates an anti-tumor immune response, is suitable for use in themethods disclosed herein.

In certain embodiments, the inhibitory immunoregulator is a component ofthe B7 family signaling pathway. In certain embodiments, the B7 familymembers are B7-H3 and B7-H4. Accordingly, certain embodiments of thedisclosure provide methods for immunotherapy of a subject afflicted withcancer, which methods comprise administering to the subject atherapeutically effective amount of an antibody or an antigen-bindingportion thereof that targets B7-H3 or H4. The B7 family does not haveany defined receptors but these ligands are upregulated on tumor cellsor tumor-infiltrating cells. Preclinical mouse models have shown thatblockade of these ligands can enhance anti-tumor immunity. An exemplaryantibody that targets B7-H3 is MGA271 (Macrogenics), currentlyundergoing human trials. Other suitable antibodies that target LAG3 aredisclosed in U.S. Patent Application 2013/0149236, herein incorporatedby reference. It will be understood by one of ordinary skill that anyantibody which binds to B7-H3 or H4, and stimulates an anti-tumor immuneresponse, is suitable for use in the methods disclosed herein.

In certain embodiments, the inhibitory immunoregulator is a component ofthe TIM3 (T cell membrane protein 3) signaling pathway. Accordingly,certain embodiments of the disclosure provide methods for immunotherapyof a subject afflicted with cancer, which methods comprise administeringto the subject a therapeutically effective amount of an antibody or anantigen-binding portion thereof that targets LAG3 and disrupts itsinteraction with galectin 9. Suitable antibodies that target TIM3 aredisclosed in U.S. Patent Application 2013/0022623, herein incorporatedby reference. It will be understood by one of ordinary skill that anyantibody which binds to TIM3, disrupts its interaction with galectin 9,and stimulates an anti-tumor immune response, is suitable for use in themethods disclosed herein.

It should be understood that antibodies targeting immune checkpointssuitable for use in the methods disclosed herein are not limited tothose described supra. Moreover, it will be understood by one ofordinary skill in the art that other immune checkpoint targets can alsobe targeted by antagonists or antibodies in the methods describedherein, provided that the targeting results in the stimulation of ananti-tumor immune response as reflected in, e.g., an increase in T cellproliferation, enhanced T cell activation, and/or increased cytokineproduction (e.g., IFN-γ, IL-2).

IV. Cancer Vaccine

In some aspects, the disclosure provides immunomodulatory fusionproteins to be used or performed in conjunction with a cancer vaccine.In certain embodiments, the cancer vaccine stimulates a specific immuneresponse against a specific target, such as a tumor-associated antigen.

In certain embodiments, the cancer vaccine includes viral, bacterial oryeast vectors to deliver recombinant genes to antigen presenting cells(APCs).

In certain embodiments the cancer vaccine uses autologous or allogeneictumor cells. In certain embodiments, these tumor cells may be modifiedfor expression of MHC, costimulatory molecules, or cytokines.

In certain embodiments, the tumor-associated antigen is determined bysequencing a patient's tumor cells and identifying mutated proteins onlyfound in the tumor. These antigens are referred to as “neoantigens.”Once a neoantigen has been identified, it can be used as the antigen fora vaccine or for developing monoclonal antibodies specifically reactivewith the neoantigen.

In certain embodiments, the vaccine includes irradiated tumor cellstransduced with cytokines such as GM-CSF or loaded with adjuvantcompounds, such as the GM-CSF-secreting tumor cell vaccine GVAX(Immunological Reviews, 222(1): 287-298, 2008). In certain embodimentsthe vaccine includes one or more tumor-associated antigens in the formof an immunogenic composition, optionally in combination with anadjuvant. For example, vaccination against HPV-16 oncoproteins resultedin positive clinical outcomes for vulvar intraepithelial neoplasia (TheNew England Journal of Medicine, 361(19), 1838-1847, 2012). Also,multipeptide immune response to cancer vaccine IMA901 after single-dosecyclophosphamide associates with longer patient survival (NatureMedicine, 18(8): 1254-61, 2012). Alternatively, a DNA-based approach canbe used to immunize a patient with one or more tumor-associatedantigens. Improved tumor immunity is observed using a DNA vaccine incombination with an anti-tyrosinase related protein-1 monoclonalantibody in murine melanoma (Cancer Research, 68(23), 9884-9891, 2008).

Other vaccine approaches utilize patient immune cells, such as dendriticcells which can be cultured with a tumor-associated antigen to produceantigen presenting cells that will stimulate the immune system andtarget the antigen of interest. A current FDA approved cancer treatmentvaccine using this approach is Provenge® (Dendreon), approved for use insome men with metastatic prostate cancer. This vaccine stimulates animmune response to prostatic acid phosphatase (PAP), an antigen found onmost prostate cancer cells. The vaccine is created by isolating aspecific patient's immune cells and culturing dendritic cells with PAPto produce antigen presenting cells that will stimulate the immunesystem and target PAP. These and other cancer vaccines can be used incombination with other treatments as described herein.

A. Amphiphile Vaccines

In some embodiments, the cancer vaccine suitable for use with theimmunomodulatory fusion proteins described herein is an amphiphilevaccine, as described in US 2013/0295129, herein incorporated byreference. An amphiphile vaccine combines an albumin-binding lipid and apeptide antigen or molecular adjuvant to efficiently target the peptideor adjuvant to lymph nodes in vivo. Lipid conjugates bind to endogenousalbumin, which targets them to lymphatics and draining lymph nodes wherethey accumulate due to the filtering of albumin by antigen presentingcells. When the lipid conjugate includes an antigenic peptide ormolecular adjuvant, the conjugates induce or enhance a robust immuneresponse.

Lymph node-targeting conjugates typically include three domains: ahighly lipophilic, albumin-binding domain (e.g., an albumin-bindinglipid), a cargo such as a molecular adjuvant or a peptide antigen, and apolar block linker, which promotes solubility of the conjugate andreduces the ability of the lipid to insert into cellular plasmamembranes. Accordingly, in certain embodiments, the general structure ofthe conjugate is L-P-C, where “L” is an albumin-binding lipid, “P” is apolar block, and “C” is a cargo such as a molecular adjuvant or apolypeptide. In some embodiments, the cargo itself can also serve as thepolar block domain, and a separate polar block domain is not required.Therefore, in certain embodiments the conjugate has only two domains: analbumin-binding lipid and a cargo.

The cargo of the conjugates suitable for use in the methods disclosedherein is typically a molecular adjuvant such as an immunostimulatoryoligonucleotide, or a peptide antigen. However, the cargo can also beother oligonucleotides, peptides, Toll-like receptor agonists or otherimmunomodulatory compounds, dyes, MRI contrast agents, fluorophores orsmall molecule drugs that require efficient trafficking to the lymphnodes.

In certain embodiments, a lipid-oligonucleotide conjugates includes animmunostimulatory oligonucleotide which is conjugated directly to alipid, or is linked to a linker which is conjugated to a lipid. Otherembodiments are directed to lipid-peptide conjugates which include anantigenic peptide conjugated directly to a lipid, or is linked to alinker which is conjugated to a lipid.

Lipids

The lipid conjugates typically include a hydrophobic lipid. The lipidcan be linear, branched, or cyclic. The lipid is preferably at least 17to 18 carbons in length, but may be shorter if it shows good albuminbinding and adequate targeting to the lymph nodes. Lymph node-targetingconjugates include lipid-oligonucleotide conjugates and lipid-peptideconjugates that can be trafficked from the site of delivery through thelymph to the lymph node. In certain embodiments, the activity relies,in-part, on the ability of the conjugate to associate with albumin inthe blood of the subject. Therefore, lymph node-targeted conjugatestypically include a lipid that can bind to albumin under physiologicalconditions. Lipids suitable for targeting the lymph node can be selectedbased on the ability of the lipid or a lipid conjugate including thelipid to bind to albumin. Suitable methods for testing the ability ofthe lipid or lipid conjugate to bind to albumin are known in the art.

For example, in certain embodiments, a plurality of lipid conjugates isallowed to spontaneously form micelles in aqueous solution. The micellesare incubated with albumin, or a solution including albumin such asFetal Bovine Serum (FBS). Samples can be analyzed, for example, byELISA, size exclusion chromatography or other methods to determine ifbinding has occurred. Lipid conjugates can be selected as lymphnode-targeting conjugates if in the presence of albumin, or a solutionincluding albumin such as Fetal Bovine Serum (FBS), the micellesdissociate and the lipid conjugates bind to albumin as discussed above.

Examples of preferred lipids for use in lymph node targeting lipidconjugates include, but are not limited to, fatty acids with aliphatictails of 8-30 carbons including, but not limited to, linear unsaturatedand saturated fatty acids, branched saturated and unsaturated fattyacids, and fatty acids derivatives, such as fatty acid esters, fattyacid amides, and fatty acid thioesters, diacyl lipids, cholesterol,cholesterol derivatives, and steroid acids such as bile acids, Lipid Aor combinations thereof.

In certain embodiments, the lipid is a diacyl lipid or two-tailed lipid.In some embodiments, the tails in the diacyl lipid contain from about 8to about 30 carbons and can be saturated, unsaturated, or combinationsthereof. The tails can be coupled to the head group via ester bondlinkages, amide bond linkages, thioester bond linkages, or combinationsthereof. In a particular embodiment, the diacyl lipids are phosphatelipids, glycolipids, sphingolipids, or combinations thereof.

Preferably, lymph node-targeting conjugates include a lipid that is 8 ormore carbon units in length. It is believed that increasing the numberof lipid units can reduce insertion of the lipid into plasma membrane ofcells, allowing the lipid conjugate to remain free to bind albumin andtraffic to the lymph node.

For example, the lipid can be a diacyl lipid composed of two C18hydrocarbon tails. In certain embodiments, the lipid for use inpreparing lymph node targeting lipid conjugates is not a single chainhydrocarbon (e.g., C18), or cholesterol. Cholesterol conjugation hasbeen explored to enhance the immunomodulation of molecular adjuvantssuch as CpG and immunogenicity of peptides, but cholesterol conjugates,which associate well with lipoproteins but poorly with albumin, showpoor lymph node targeting and low immunogenicity in vaccines compared tooptimal albumin-binding conjugates.

Molecular Adjuvants

In certain embodiments, lipid-oligonucleotide conjugates are used in thevaccine. The oligonucleotide conjugates typically contain animmunostimulatory oligonucleotide.

In certain embodiments, the immunostimulatory oligonucleotide can serveas a ligand for pattern recognition receptors (PRRs). Examples of PRRsinclude the Toll-like family of signaling molecules that play a role inthe initiation of innate immune responses and also influence the laterand more antigen specific adaptive immune responses. Therefore, theoligonucleotide can serve as a ligand for a Toll-like family signalingmolecule, such as Toll-Like Receptor 9 (TLR9).

For example, unmethylated CpG sites can be detected by TLR9 onplasmacytoid dendritic cells and B cells in humans (Zaida, et al.,Infection and Immunity, 76(5):2123-2129, (2008)). Therefore, thesequence of oligonucleotide can include one or more unmethylatedcytosine-guanine (CG or CpG, used interchangeably) dinucleotide motifs.The ‘p’ refers to the phosphodiester backbone of DNA, as discussed inmore detail below, some oligonucleotides including CG can have amodified backbone, for example a phosphorothioate (PS) backbone. Incertain embodiments, an immunostimulatory oligonucleotide can containmore than one CG dinucleotide, arranged either contiguously or separatedby intervening nucleotide(s). The CpG motif(s) can be in the interior ofthe oligonucleotide sequence. Numerous nucleotide sequences stimulateTLR9 with variations in the number and location of CG dinucleotide(s),as well as the precise base sequences flanking the CG dimers.

Typically, CG ODNs are classified based on their sequence, secondarystructures, and effect on human peripheral blood mononuclear cells(PBMCs). The five classes are Class A (Type D), Class B (Type K), ClassC, Class P, and Class S (Vollmer, J & Krieg, A M, Advanced drug deliveryreviews 61(3): 195-204 (2009), incorporated herein by reference). CGODNs can stimulate the production of Type I interferons (e.g., IFNα) andinduce the maturation of dendritic cells (DCs). Some classes of ODNs arealso strong activators of natural killer (NK) cells through indirectcytokine signaling. Some classes are strong stimulators of human B celland monocyte maturation (Weiner, G L, PNAS USA 94(20): 10833-7 (1997);Dalpke, A H, Immunology 106(1): 102-12 (2002); Hartmann, G, J of Immun.164(3):1617-2 (2000), each of which is incorporated herein byreference).

According to some embodiments, a lipophilic-CpG oligonucleotideconjugate is used to enhance an immune response to a peptide antigen.The lipophilic-CpG oligonucleotide is represented by the following,wherein “L” is a lipophilic compound, such as diacyl lipid, “Gn” is aguanine repeat linker and “n” represents 1, 2, 3, 4, or 5.

(SEQ ID NO: 205) 5′-L-G_(n)TCCATGACGTTCCTGACGTT-3′

Other PRR Toll-like receptors include TLR3, and TLR7 which may recognizedouble-stranded RNA, single-stranded and short double-stranded RNAs,respectively, and retinoic acid-inducible gene I (RIG-I)-like receptors,namely RIG-I and melanoma differentiation-associated gene 5 (MDA5),which are best known as RNA-sensing receptors in the cytosol. Therefore,in certain embodiments, the oligonucleotide contains a functional ligandfor TLR3, TLR7, or RIG-I-like receptors, or combinations thereof.

Examples of immunostimulatory oligonucleotides, and methods of makingthem are known in the art, see for example, Bodera, P. Recent PatInflamm Allergy Drug Discov. 5(1):87-93 (2011), incorporated herein byreference.

In certain embodiments, the oligonucleotide cargo includes two or moreimmunostimulatory sequences.

The oligonucleotide can be between 2-100 nucleotide bases in length,including for example, 5 nucleotide bases in length, 10 nucleotide basesin length, 15 nucleotide bases in length, 20 nucleotide bases in length,25 nucleotide bases in length, 30 nucleotide bases in length, 35nucleotide bases in length, 40 nucleotide bases in length, 45 nucleotidebases in length, 50 nucleotide bases in length, 60 nucleotide bases inlength, 70 nucleotide bases in length, 80 nucleotide bases in length, 90nucleotide bases in length, 95 nucleotide bases in length, 98 nucleotidebases in length, 100 nucleotide bases in length or more.

The 3′ end or the 5′ end of the oligonucleotides can be conjugated tothe polar block or the lipid. In certain embodiments the 5′ end of theoligonucleotide is linked to the polar block or the lipid.

The oligonucleotides can be DNA or RNA nucleotides which typicallyinclude a heterocyclic base (nucleic acid base), a sugar moiety attachedto the heterocyclic base, and a phosphate moiety which esterifies ahydroxyl function of the sugar moiety. The principal naturally-occurringnucleotides comprise uracil, thymine, cytosine, adenine and guanine asthe heterocyclic bases, and ribose or deoxyribose sugar linked byphosphodiester bonds. In certain embodiments, the oligonucleotides arecomposed of nucleotide analogs that have been chemically modified toimprove stability, half-life, or specificity or affinity for a targetreceptor, relative to a DNA or RNA counterpart. The chemicalmodifications include chemical modification of nucleobases, sugarmoieties, nucleotide linkages, or combinations thereof. As used herein‘modified nucleotide” or “chemically modified nucleotide” defines anucleotide that has a chemical modification of one or more of theheterocyclic base, sugar moiety or phosphate moiety constituents. Incertain embodiments, the charge of the modified nucleotide is reducedcompared to DNA or RNA oligonucleotides of the same nucleobase sequence.For example, the oligonucleotide can have low negative charge, nocharge, or positive charge.

Typically, nucleoside analogs support bases capable of hydrogen bondingby Watson-Crick base pairing to standard polynucleotide bases, where theanalog backbone presents the bases in a manner to permit such hydrogenbonding in a sequence-specific fashion between the oligonucleotideanalog molecule and bases in a standard polynucleotide (e.g.,single-stranded RNA or single-stranded DNA). In certain embodiments, theanalogs have a substantially uncharged, phosphorus containing backbone.

Peptide Antigens

The peptide conjugates suitable for use in the methods disclosed hereintypically include an antigenic protein or polypeptide, such as atumor-associated antigen or portion thereof.

The peptide can be 2-100 amino acids, including for example, 5 aminoacids, 10 amino acids, 15 amino acids, 20 amino acids, 25 amino acids,30 amino acids, 35 amino acids, 40 amino acids, 45 amino acids, or 50amino acids. In some embodiments, a peptide can be greater than 50 aminoacids. In some embodiments, the peptide can be >100 amino acids. Aprotein/peptide can be linear, branched or cyclic. The peptide caninclude D amino acids, L amino acids, or a combination thereof. Thepeptide or protein can be conjugated to the polar block or lipid at theN-terminus or the C-terminus of the peptide or protein.

The protein or polypeptide can be any protein or peptide that can induceor increase the ability of the immune system to develop antibodies andT-cell responses to the protein or peptide. A cancer antigen is anantigen that is typically expressed preferentially by cancer cells(i.e., it is expressed at higher levels in cancer cells than onnon-cancer cells) and in some instances it is expressed solely by cancercells. The cancer antigen may be expressed within a cancer cell or onthe surface of the cancer cell. The cancer antigen can be, but is notlimited to, TRP-1, TRP-2, MART-1/Melan-A, gp100, adenosinedeaminase-binding protein (ADAbp), FAP, cyclophilin b, colorectalassociated antigen (CRC)-C017-1A/GA733, carcinoembryonic antigen (CEA),CAP-1, CAP-2, etv6, AML1, prostate specific antigen (PSA), PSA-1, PSA-2,PSA-3, prostate-specific membrane antigen (PSMA), T cellreceptor/CD3-zeta chain, and CD20. The cancer antigen may be selectedfrom the group consisting of MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4,MAGE-A5, MAGE-A6, MAGE-A7, MAGE-A8, MAGE-A9, MAGE-A10, MAGE-A11,MAGE-A12, MAGE-Xp2 (MAGE-B2), MAGE-Xp3 (MAGE-B3), MAGE-Xp4 (MAGE-B4),MAGE-C1, MAGE-C2, MAGE-C3, MAGE-C4, MAGE-05), GAGE-1, GAGE-2, GAGE-3,GAGE-4, GAGE-5, GAGE-6, GAGE-7, GAGE-8, GAGE-9, BAGE, RAGE, LAGE-1, NAG,GnT-V, MUM-1, CDK4, tyrosinase, p53, MUC family, HER2/neu, p21ras,RCAS1, α-fetoprotein, E-cadherin, α-catenin, β-catenin, γ-catenin,p120ctn, gp100Pmel 17, PRAME, NY-ESO-1, cdc27, adenomatous polyposiscoli protein (APC), fodrin, Connexin 37, Ig-idiotype, p15, gp75, GM2ganglioside, GD2 ganglioside, human papilloma virus proteins, Smadfamily of tumor antigens, Imp-1, P1A, EBV-encoded nuclear antigen(EBNA)-1, brain glycogen phosphorylase, SSX-1, SSX-2 (HOM-MEL-40),SSX-1, SSX-4, SSX-5, SCP-1 and CT-7, CD20, or c-erbB-2. Additionalcancer antigens include the tumor antigens described herein.

Suitable antigens are known in the art and are available from commercialgovernment and scientific sources. In certain embodiments, the antigensare whole inactivated or irradiated tumor cells. The antigens may bepurified or partially purified polypeptides derived from tumors. Theantigens can be recombinant polypeptides produced by expressing DNAencoding the polypeptide antigen in a heterologous expression system.The antigens can be DNA encoding all or part of an antigenic protein.The DNA may be in the form of vector DNA such as plasmid DNA.

In certain embodiments, antigens may be provided as single antigens ormay be provided in combination. Antigens may also be provided as complexmixtures of polypeptides or nucleic acids.

Polar Block/Linker

For the conjugate to be trafficked efficiently to the lymph node, theconjugate should remain soluble. Therefore, a polar block linker can beincluded between the cargo and the lipid to increase solubility of theconjugate. The polar block reduces or prevents the ability of the lipidto insert into the plasma membrane of cells, such as cells in the tissueadjacent to the injection site. The polar block can also reduce orprevent the ability of cargo, such as synthetic oligonucleotidescontaining a PS backbone, from non-specifically associating withextracellular matrix proteins at the site of administration. The polarblock increases the solubility of the conjugate without preventing itsability to bind to albumin. It is believed that this combination ofcharacteristics allows the conjugate to bind to albumin present in theserum or interstitial fluid, and remain in circulation until the albuminis trafficked to, and retained in a lymph node. The length andcomposition of the polar block can be adjusted based on the lipid andcargo selected. For example, for oligonucleotide conjugates, theoligonucleotide itself may be polar enough to insure solubility of theconjugate, for example, oligonucleotides that are 10, 15, 20 or morenucleotides in length. Therefore, in certain embodiments, no additionalpolar block linker is required. However, depending on the amino acidsequence, some lipidated peptides can be essentially insoluble. In thesecases, it can be desirable to include a polar block that mimics theeffect of a polar oligonucleotide.

A polar block can be used as part of any of lipid conjugates suitablefor use in the methods disclosed herein, for example,lipid-oligonucleotide conjugates and lipid-peptide conjugates, whichreduce cell membrane insertion/preferential portioning ont albumin.Suitable polar blocks include, but are not limited to, oligonucleotidessuch as those discussed above, a hydrophilic polymer including but notlimited to poly(ethylene glycol) (MW: 500 Da to 20,000 Da),polyacrylamide (MW: 500 Da to 20,000 Da), polyacrylic acid; a string ofhydrophilic amino acids such as serine, threonine, cysteine, tyrosine,asparagine, glutamine, aspartic acid, glutamic acid, lysine, arginine,histidine, or combinations thereof polysaccharides, including but notlimited to, dextran (MW: 1,000 Da to 2,000,000 Da), or combinationsthereof.

The hydrophobic lipid and the linker/cargo are covalently linked. Thecovalent bond may be a non-cleavable linkage or a cleavable linkage. Thenon-cleavable linkage can include an amide bond or phosphate bond, andthe cleavable linkage can include a disulfide bond, acid-cleavablelinkage, ester bond, anhydride bond, biodegradable bond, orenzyme-cleavable linkage.

a. Ethylene Glycol Linkers

In certain embodiments, the polar block is one or more ethylene glycol(EG) units, more preferably two or more EG units (i.e., polyethyleneglycol (PEG)). For example, in certain embodiments, a peptide conjugateincludes a protein or peptide (e.g., peptide antigen) and a hydrophobiclipid linked by a polyethylene glycol (PEG) molecule or a derivative oranalog thereof.

In certain embodiments, protein conjugates suitable for use in themethods disclosed herein contain protein antigen linked to PEG which isin turn linked to a hydrophobic lipid, or lipid-Gn-ON conjugates, eithercovalently or via formation of protein-oligo conjugates that hybridizeto oligo micelles. The precise number of EG units depends on the lipidand the cargo, however, typically, a polar block can have between about1 and about 100, between about 20 and about 80, between about 30 andabout 70, or between about 40 and about 60 EG units. In certainembodiments, the polar block has between about 45 and 55 EG, units. Forexample, in certain embodiments, the polar block has 48 EG units.

b. Oligonucleotide Linkers

As discussed above, in certain embodiments, the polar block is anoligonucleotide. The polar block linker can have any sequence, forexample, the sequence of the oligonucleotide can be a random sequence,or a sequence specifically chosen for its molecular or biochemicalproperties (e.g., highly polar). In certain embodiments, the polar blocklinker includes one or more series of consecutive adenine (A), cytosine(C), guanine (G), thymine (T), uracil (U), or analog thereof. In certainembodiments, the polar block linker consists of a series of consecutiveadenine (A), cytosine (C), guanine (G), thymine (T), uracil (U), oranalog thereof.

In certain embodiments, the linker is one or more guanines, for examplebetween 1-10 guanines. It has been discovered that altering the numberof guanines between a cargo such as a CpG oligonucleotide, and a lipidtail controls micelle stability in the presence of serum proteins.Therefore, the number of guanines in the linker can be selected based onthe desired affinity of the conjugate for serum proteins such asalbumin. When the cargo is a CpG immunostimulatory oligonucleotide andthe lipid tail is a diacyl lipid, the number of guanines affects theability of micelles formed in aqueous solution to dissociate in thepresence of serum: 20% of the non-stabilized micelles(lipo-G₀T₁₀-CG)(SEQ ID NO: 206) were intact, while the remaining 80%were disrupted and bonded with FBS components. In the presence ofguanines, the percentage of intact micelles increased from 36%(lipo-G₂T₈-CG) (SEQ ID NO: 207) to 73% (lipo-G₄T₆-CG) (SEQ ID NO: 208),and finally reached 90% (lipo-G₆T₄-CG) (SEQ ID NO: 209). Increasing thenumber of guanines to eight (lipo-G₈T₂-CG) (SEQ ID NO: 210) and ten(lipo-G₁₀T₀-CG) (SEQ ID NO: 211) did not further enhance micellestability.

Therefore, in certain embodiments, the linker in a lymph node-targetingconjugate suitable for use in the methods disclosed herein can include0, 1, or 2 guanines. As discussed in more detail below, linkers thatinclude 3 or more consecutive guanines can be used to formmicelle-stabilizing conjugates with properties that are suitable for usein the methods disclosed herein.

B. Immunogenic Compositions

The conjugates suitable for use in the methods disclosed herein can beused in immunogenic compositions or as components in vaccines.Typically, immunogenic compositions disclosed herein include anadjuvant, an antigen, or a combination thereof. The combination of anadjuvant and an antigen can be referred to as a vaccine. Whenadministered to a subject in combination, the adjuvant and antigen canbe administered in separate pharmaceutical compositions, or they can beadministered together in the same pharmaceutical composition. Whenadministered in combination, the adjuvant can be a lipid conjugate, theantigen can be a lipid conjugate, or the adjuvant and the antigen canboth be lipid conjugates.

An immunogenic composition suitable for use in the methods disclosedherein can include a lipid conjugate that is an antigen such as anantigenic polypeptide-lipid conjugate, administered alone, or incombination with an adjuvant. The adjuvant may be without limitationalum (e.g., aluminum hydroxide, aluminum phosphate); saponins purifiedfrom the bark of the Q. saponaria tree such as QS21 (a glycolipid thatelutes in the 21st peak with HPLC fractionation; Antigenics, Inc.,Worcester, Mass.); poly[di(carboxylatophenoxy)phosphazene (PCPP polymer;Virus Research Institute, USA), Flt3 ligand, Leishmania elongationfactor (a purified Leishmania protein; Corixa Corporation, Seattle,Wash.), ISCOMS (immunostimulating complexes which contain mixedsaponins, lipids and form virus-sized particles with pores that can holdantigen; CSL, Melbourne, Australia), Pam3Cys, SB-AS4 (SmithKline Beechamadjuvant system #4 which contains alum and MPL; SBB, Belgium), non-ionicblock copolymers that form micelles such as CRL 1005 (these contain alinear chain of hydrophobic polyoxypropylene flanked by chains ofpolyoxyethylene, Vaxcel, Inc., Norcross, Ga.), and Montanide IMS (e.g.,IMS 1312, water-based nanoparticles combined with a solubleimmunostimulant, Seppic).

Adjuvants may be TLR ligands, such as those discussed above. Adjuvantsthat act through TLR3 include, without limitation, double-stranded RNA.Adjuvants that act through TLR4 include, without limitation, derivativesof lipopolysaccharides such as monophosphoryl lipid A (MPLA; RibiImmunoChem Research, Inc., Hamilton, Mont.) and muramyl dipeptide (MDP;Ribi) and threonyl-muramyl dipeptide (t-MDP; Ribi); OM-174 (aglucosamine disaccharide related to lipid A; OM Pharma SA, Meyrin,Switzerland). Adjuvants that act through TLR5 include, withoutlimitation, flagellin. Adjuvants that act through TLR7 and/or TLR8include single-stranded RNA, oligoribonucleotides (ORN), synthetic lowmolecular weight compounds such as imidazoquinolinamines (e.g.,imiquimod (R-837), resiquimod (R-848)). Adjuvants acting through TLR9include DNA of viral or bacterial origin, or syntheticoligodeoxynucleotides (ODN), such as CpG ODN. Another adjuvant class isphosphorothioate containing molecules such as phosphorothioatenucleotide analogs and nucleic acids containing phosphorothioatebackbone linkages.

The adjuvant can also be oil emulsions (e.g., Freund's adjuvant);saponin formulations; virosomes and viral-like particles; bacterial andmicrobial derivatives; immunostimulatory oligonucleotides;ADP-ribosylating toxins and detoxified derivatives; alum; BCG;mineral-containing compositions (e.g., mineral salts, such as aluminiumsalts and calcium salts, hydroxides, phosphates, sulfates, etc.);bioadhesives and/or mucoadhesives; microparticles; liposomes;polyoxyethylene ether and polyoxyethylene ester formulations;polyphosphazene; muramyl peptides; imidazoquinolone compounds; andsurface active substances (e.g. lysolecithin, pluronic polyols,polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, anddinitrophenol).

Adjuvants may also include immunomodulators such as cytokines,interleukins (e.g., IL-1, IL-2, IL-4, IL-5, IL-6, IL-7, IL-12, etc.),interferons (e.g., interferon-.gamma.), macrophage colony stimulatingfactor, and tumor necrosis factor.

Kits

In some aspects, the disclosure provides kits comprising at least animmunomodulatory fusion protein described herein and instructions foruse. In some embodiments, the kits comprise, in a suitable container, animmunomodulatory fusion protein, one or more controls, and variousbuffers, reagents, enzymes and other standard ingredients well known inthe art. In some embodiments, the kits further comprise instructions foruse in combination with an immunotherapy.

In some embodiments, the container is at least one vial, well, testtube, flask, bottle, syringe, or other container means, into which animmunomodulatory fusion protein may be placed, and in some instances,suitably aliquoted. When an additional component is provided, the kitcan contain additional containers into which this compound may beplaced. The kits can also include a means for containing animmunomodulatory fusion protein, and any other reagent containers inclose confinement for commercial sale. Such containers may includeinjection or blow-molded plastic containers into which the desired vialsare retained. Containers and/or kits can include labeling withinstructions for use and/or warnings.

In some embodiments, the disclosure provides a kit comprising acontainer comprising an immunomodulatory fusion protein describedherein, an optional pharmaceutically acceptable carrier, and a packageinsert comprising instructions for administration of the composition fortreating or delaying progression of cancer in an individual receiving animmunotherapy (e.g., CAR-T cells, cancer vaccine, anti-tumor associatedantigen antibody, and/or immune checkpoint blockade).

In some embodiments, the disclosure provides a kit comprising amedicament comprising an immunomodulatory fusion protein describedherein, an optional pharmaceutically acceptable carrier, and a packageinsert comprising instructions for administration of the medicamentalone or in combination with an immunotherapy (e.g., CAR-T cells, cancervaccine, anti-tumor associated antigen antibody, and/or immunecheckpoint blockade), for treating or delaying progression of cancer inan individual receiving CAR-T cell therapy.

Other Embodiments—Collagen-Binding IgG-Binding Fusion Proteins

In another aspect, the disclosure provides collagen-binding IgG-bindingfusion proteins comprising an Ig-binding domain and a collagen-bindingdomain. The collagen-binding IgG-binding fusion proteins provided by thedisclosure bind to an IgG (e.g., an immunomodulatory IgG) and tocollagen, thereby localizing or sequestering the IgG within a tumor whenadministered.

In some embodiments, the collagen-binding domain is a collagen-bindingdomain as described supra. Exemplary IgG binding domains include adimerized Z domain (one of the five IgG binding domains of protein A,herein referred to as “ZZ”) (Jendeberg et al., (1995) J Mol Recognit8:270-278), a dimerized IgG binding domain of protein G (herein referredto as “SpG2”) (Jung et al., (2009) Anal Chem 81:936-942), an IgG binderisolated from a Sso7d yeast display library (Gera et al., (2011) J MolBiol 409:601-616), an IgG binder isolated from a Fibronectin type IIIdomain (Fn3) yeast display library (Hackel et al., (2010) J Mol Biol401:84-96), and two small peptides designed to bind IgG Fc regions(herein referred to as “Fc-III-4C” and “RRGW) (Gong et al., (2015)Bioconjug Chem 27:1569-1573; Tsai et al., (2014) Anal Chem86:2931-2938).

In some embodiments, the collagen-binding IgG-binding fusion protein issuitable for use in any of the methods described herein. In someembodiments, the collagen-binding IgG-binding fusion protein is used incombination with a therapeutic antibody (e.g., an immunomodulatoryantibody). In some embodiments, the collagen-binding IgG-binding fusionprotein is administered in combination with a therapeutic antibody forthe treatment of cancer.

EXAMPLES

While the present disclosure has been described with reference to thespecific embodiments thereof, it should be understood by those skilledin the art that various changes may be made and equivalents may besubstituted without departing from the true spirit and scope of thedisclosure. In addition, many modifications may be made to adapt aparticular situation, material, composition of matter, process, processstep or steps, to the objective, spirit and scope of the presentdisclosure. All such modifications are intended to be within the scopeof the disclosure.

Example 1: Recombinant Expression of Collagen-Binding Fusion Proteins inMammalian Cells

To evaluate the ability to express collagen-binding immunomodulatorymolecules in mammalian cells, five His-tagged collagen-bindingpolypeptides fused to Gaussia Luciferase (Gluc) (collagen imaging probe(CNA35-Gluc), bacterial collagenase ColG domains s3a/s3b (ColGs3a/s3b-Gluc), heparin binding domain of murine placenta growth factor-2(PLGF2HBD-Gluc), bacterial collagenase ColH domain s3 (ColH s3-Gluc),and murine protein lumican (Lumican-Gluc)) were transiently expressed inhuman embryonic kidney 293 (HEK293) cells. The amino acid sequences forthese constructs are set forth in SEQ ID NOs: 128-132. Briefly, HEK293cells (at 1 million cells/mL density) were transfected withsterile-filtered plasmid DNA (1 mg per liter cell culture) usingpolyethylenimine (2 mg per liter cell culture) in OptiPro serum-freemedia (20 mL per liter cell culture) (Thermo Fisher). TA99 was purifiedusing rProtein A Sepharose Fast Flow resin (GE Healthcare) as previouslydescribed (Zhu et al. 2015). His-tagged proteins were isolated fromHEK293 supernatant using TALON Metal Affinity Resin (Takara Bio Inc.).Cytokine-fusion proteins were then further purified by size exclusionchromatrography using a HiLoad 16/600 Superdex 200 pg column on an AKTAFPLC system (GE Healthcare) that had been pretreated for 4 hours with 1M NaOH to remove endotoxin and subsequently equilibrated in sterile PBS(Corning). Following purification, all proteins were buffer exchangedinto sterile PBS (Corning), 0.2 micron sterile-filtered (PallCorporation) and confirmed to contain minimal levels of endotoxin (<0.1EU per injection) using an chromogenic LAL assay (Lonza). To confirmtheir molecular weights, proteins were run alongside a Novex PrestainedSharp Protein Ladder on a 4-12% NuPAGE Bis-Tris protein gel (LifeTechnologies) with 1% MES running buffer. All proteins were stored at 4°C. but, prior to therapeutic injection, cytokine fusion proteins werewarmed to room temperature to rescue lumican which demonstratesreversible cold denaturation. The relative expression levels of theHis-tagged collagen-binding fusion proteins in the resulting eluates wasevaluated by SDS-PAGE and by absorption spectrophotometry followingsize-exclusion chromatography of TALON-purification eluates.

Transient expression of Gluc (19.8 kDa) alone as well as Gluc-fusedcollagen-binding polypeptides ColG s3a/s3b-Gluc (46.1 kDa), ColH s3-Gluc(32.8 kDa), and Lumican-Gluc (56.6 kDa) in HEK293 cells was achieved, asdetermined by SDS-PAGE analysis of His-tagged proteins purified from HEKcell lysates. Protein staining at or near the respective expectedmolecular weight of each fusion protein was observed (data not shown).However, no expression was observed for CNA35-Gluc (54.4 kDa) orPLGF2HBD-Gluc (22.8 kDa) as measured by SDS-PAGE of protein purifiedfrom HEK cell lysates (data not shown).

Transient expression of Gluc, ColG s3a/s3b-Gluc, ColH s3-Gluc, andLumican-Gluc in HEK293 cells was achieved, as determined by absorptionspectrophotometry during size exclusion chromatography of recombinantlyexpressed and purified His-tagged proteins. A monomeric peak of UVradiation absorption at 280 nm (A280) was observed for each fusionprotein (data not shown). No absorbance peaks were detected forCNA35-Gluc or PLGF2HBD-Gluc.

In addition to lumican, other mammalian collagen-binding polypeptidescan be expressed as collagen-binding fusion proteins. The extracellulardomain of the mammalian leukocyte-associated immunoglobulin-likereceptor 1 (LAIR-1) protein is known to bind collagen (Lebbink et al.,(2006) J Exp Med 203(6):1419-1425). Transient expression of theextracellular domain of His-tagged murine LAIR-1 (amino acid sequenceset forth in SEQ ID NO: 181) in HEK293 as described above was achieved,as determined by absorption spectrophotometry during size exclusionchromatography of recombinantly expressed protein. LAIR-1 eluted as amonomeric peak as measured by UV radiation absorption at 280 nm (A280)(data not shown).

These results demonstrate that collagen-binding fusion proteinscomprising prokaryotic or mammalian collagen-binding polypeptidesexpress in mammalian cells. Expression of the His-taggedcollagen-binding fusion proteins ColG s3a/s3b-Gluc, ColH s3-Gluc, andLumican-Gluc as well as Gluc alone in HEK293 cells was achieved, whileexpression of CNA35-Gluc or PLGF2HBD-Gluc was not observed. Further, theextracellular domain of His-tagged LAIR-1 in HEK293 cells was expressedand purified. These results suggest that collagen-bindingimmunomodulatory molecules comprising a prokaryotic or mammaliancollagen-binding polypeptide (e.g., ColG s3a/s3b, ColH s3, Lumican orLAIR) will express in mammalian cells.

Example 2: Recombinant Collagen-Binding Fusion Proteins Bind Collagen InVitro

To evaluate the ability of collagen-binding immunomodulatory moleculesto bind collagen, the collagen-binding fusion proteins expressed andpurified as described in Example 1 were tested for their ability to bindto collagen I- and collagen IV-coated plates by ELISA. Briefly, collagenI (Gibco) and collagen IV (Corning) coated 96 well-plates were blockedat room temperature for 1 hour with PBS+0.1% wt/vol bovine serum albumin(BSA)+0.05% wt/vol Tween-20 (PBSTA) and then incubated with variousconcentrations of lumican in PBSTA for 3 hours at room temperature. Thelumican had been prewarmed for 10 minutes at 37 C to reverse itsdenaturation mediated by cold temperatures. Wells were washed with PBSTAand then incubated with a horseradish peroxidase-conjugated polyclonalanti-6×His (ab1187, Abcam) at a 1:4000 dilution in PBSTA for 1 hour atroom temperature. Wells were washed again with PBSTA and then 1-StepUltra TMB-ELISA Substrate Solution (Thermo Fisher Scientific) was addedfor 10 mins followed by 1 M sulfuric acid to stop the chromogenicreaction. Absorbance at 450 nm (corrected with a reference absorbance at570 nm) measured using an Infinite M1000 microplate reader (Tecan).Wells with titrations of MSA served as a negative control.

Purified His-tagged collagen-binding fusion proteins Lumican-Gluc, ColGs3a/s3b-Gluc, and ColH s3-Gluc were evaluated by ELISA on a collagen Icoated plate. As shown in FIG. 1A, only Lumican-Gluc and ColGs3a/s3b-Gluc bound to collagen I with a KD of 130 nM and 139 nM,respectively. ColH s3-Gluc did not bind collagen I specifically overGluc background binding.

Purified His-tagged collagen-binding fusion proteins Lumican-Gluc, ColGs3a/s3b-Gluc, and ColH s3-Gluc were evaluated by ELISA on a collagen IVcoated plate. As shown in FIG. 1B, Lumican-Gluc bound to collagen IVwith a KD of 600 nM. ColG s3a/s3b-Gluc or ColH s3-Gluc did not bindcollagen IV specifically over Gluc background binding.

Purified His-tagged murine LAIR-1 (denoted as mLAIR1-His) and His-taggedbiotinylated lumican (denoted Lwt-His-b) were evaluated for binding tocollagen type I by ELISA as a function of concentration. Binding wasdetermined by ELISA using an anti-His antibody conjugated to horseradishperoxidase (HRP). As shown in FIG. 1C, LAIR-1 and lumican had similarbinding affinity to collagen type I. Binding to a plate blocked withbovine serum albumin was also evaluated by ELISA. No binding wasobserved for either protein, indicating that measured binding tocollagen type I was specific.

To further demonstrate the collagen-binding activity of the mammaliancollagen-binding polypeptides described above, the ability of thepurified His-tagged LAIR-1 and lumican collagen-binding polypeptidesdescribed in Example 1 to competitively bind collagen was tested.Briefly, collagen I (Gibco) 96 well-plates was blocked at roomtemperature for 1 hour with PBS+0.1% wt/vol bovine serum albumin(BSA)+0.05% wt/vol Tween-20 (PBSTA) and then incubated with variousconcentrations of LAIR in the presence of 50 nM of biotinylated lumicanin PBSTA for 3 hours at room temperature. The lumican and LAIR had beenprewarmed for 10 minutes at 37 C to reverse its denaturation mediated bycold temperatures. Wells were washed with PBSTA and then incubated witha horseradish peroxidase-conjugated polyclonal Streptavidin-HRP at a1:400 dilution in PBSTA for 1 hour at room temperature. Wells werewashed again with PBSTA and then 1-Step Ultra TMB-ELISA SubstrateSolution (Thermo Fisher Scientific) was added for 10 mins followed by 1M sulfuric acid to stop the chromogenic reaction. Absorbance at 450 nm(corrected with a reference absorbance at 570 nm) measured using anInfinite M1000 microplate reader (Tecan). Wells with titrations of MSAserved as a negative control. The results of a competition ELISAdetecting lumican binding in the presence of varying concentration ofLAIR on a collagen coated plate is shown in FIG. 1D.

To evaluate the stability of a collagen-binding polypeptide followingpurification, the collagen-binding activity of lumican after thawingfrom a frozen state in solution was tested. Briefly, collagen I Affinity(K_(D)) of lumican incubated with various excipients (trehalose (T), BSA(B), collagen (C), protein alone (P)) in different conditions afterthawing from frozen: 37 C for 3 weeks (blue), 4 C for 3 weeks (gray), 4C for 2 weeks followed by 37 for 1 week (red). The binding affinity oflumican, in the presence of excipient (trehalose, bovine serum albumin,and collagen), to collagen I after thawing from frozen was determined byELISA as described previously. The binding of affinity (K_(D)) remainedsimilar regardless of which excipient was used. However, the bindingaffinity was reduced by approximately 50-fold if lumican was not warmedto 37° C. prior to measurement (data not shown).

These results demonstrate that collagen-binding fusion proteinscomprising prokaryotic (e.g., ColG s3a/s3b) or mammalian (e.g., lumican)collagen-binding polypeptides bind to collagen I (FIG. 1A). Theseresults also demonstrate that a collagen-binding fusion proteincomprising a mammalian collagen-binding polypeptide (e.g., lumican)binds to both collagen I and IV (FIG. 1B). Further, these resultsdemonstrate that the extracellular domain of LAIR-1 and lumican competefor binding to collagen I (FIG. 1C, 1D). These results suggest thatcollagen-binding immunomodulatory molecules comprising prokaryotic ormammalian collagen-binding polypeptides (e.g., ColG s3a/s3b, lumican,and LAIR-1) will bind to collagen.

Example 3: Recombinant Collagen-Binding Fusion Proteins are RetainedFollowing Intratumoral Injection

Collagens type I and IV are components of the thick fibrotic capsulesurrounding tumors and the perivascular basement membrane, respectively.To evaluate expression of collagen types I and IV in mouse tumors, micewere inoculated with 1 million 4T1, MC38 or B16F10 tumor cells on day 0.Tumors were excised on day 10 and fixed in 10% neutral buffered formalinovernight, then embedded in paraffin and section to 5 micron thickness(CryoStar NX70). The tumor cross-sections were assessed for presence ofcollagen types I and IV by immunohistochemistry. Briefly, sections werestained with rabbit antibodies against collagen type I (ab34710, Abcam)and collagen type IV (ab6586, Abcam) at a 1:500 dilution inTris-buffered saline with 0.1% vol/vol Tween-20, followed by a secondarystaining using goat HRP-conjugated anti-rabbit antibody (ab6721, Abcam).Cross-sections of 4T1 (left), MC38 (middle), and B16F10 (right) tumorsshowed positive staining for collagen type I (top) and collagen type IV(bottom) (data not shown). Thus, collagen types I and IV are abundantlyexpressed in several syngeneic murine tumor models.

To evaluate the intratumoral retention of collagen-bindingimmunomodulatory molecules, the collagen-binding fusion proteins thatbound to collagen I in Example 2 (Lumican-Gluc and ColG s3a/s3b-Gluc)were tested for their ability to remain associated at the site ofintratumoral injection by in vivo fluorescence imaging. Briefly, 5×10⁵4T1 cells (murine mammary carcinoma cells) were injected into themammary fat pad of BALB/c mice followed by intratumoral injection ofGluc alone, GolG s3a/s3b-Gluc, or Lumican-Gluc on day 7 post-tumor cellinjection. Immediately after intratumoral injection, each mouse wasmonitored by in vivo bioluminescence imaging (epi-illumination,autoexposure settings).

The bioluminescent signal from the mouse injected Gluc alone decreasedto a background level approximately 36 hrs. post injection (data notshown). The signal from the mouse injected with ColG s3a/s3b-Glucdecreased to a background level approximately 8.5 days andpost-injection (data not shown). The signal from the mouse injected withLumican-Gluc did not decrease below background level for the duration ofthe experiment (approximately 16.5 days post injection, data not shown).

These results demonstrate that the collagen-binding fusion proteins ColGs3a/s3b-Gluc and Lumican-Gluc are physically retained at the site ofintratumoral injection over time. These results suggest thatcollagen-binding immunomodulatory molecules comprising a prokaryotic ormammalian collagen-binding polypeptide (e.g., ColG s3a/s3b or Lumican)will exhibit intratumoral retention and limited systemic dissemination.

Example 4: Intratumoral Retention of Collagen-Binding Fusion ProteinsDepend on Molecular Weight and Collagen Binding Activity

Several factors may dictate the intratumoral retention of acollagen-binding fusion protein: affinity for collagen, collagenconcentration, size-dependent escape by diffusion or convection, andprotein turnover. As a protein under 60 kDa, lumican (37 kDa) faces highpermeability across vascular endothelium and is vulnerable to absorptioninto circulation, which may contribute to distribution away from theinjection site and a decrease in intratumoral retention (McLennan etal., (2005) Drug Discov Today Technol 2:89-96; Egawa et al., (2013) SciRep 3:1932).

To evaluate the effect of molecular weight on the intratumoral retentionand systemic distribution of collagen-binding immunomodulatorymolecules, the retention of the collagen-binding polypeptide lumicanfused to the 67 kDa mouse serum albumin (MSA) protein after intratumoralinjection was determined by in vivo fluorescence imaging. Briefly, fortumor inoculation of B16F10-Trp2KO, 10⁶ cells resuspended in 50 uL ofsterile PBS were injected subcutaneously into the right flanks ofC57BL/6 female mice. Fusion proteins were labeled with five-molar excessof Alexa Fluor 647 NHS Ester (Life Technologies) for 30 minutes in PBSadjusted to pH 8. Excess dye was removed using a PD10 Desalting Column(GE Healthcare) and degree of labeling (DOL) for each proteincalculated. Proteins compared in retention studies contained equimolaramount of dye. For Lumican-MSA (SEQ ID NO: 126) and MSA (SEQ ID NO:183), 0.11 nmol of each construct (110 ug of Lumican-MSA and 71.7 ug ofMSA was administered) was injected intratumorally into mice bearingB16F10-Trp2KO tumors five days after inoculation.

For assessing fusion protein retention, mice were imaged with a XenogenIVIS Imaging System 100 (Xenogen) at indicated time points underauto-exposure epi-illumination fluorescence settings. During this time,mice were maintained on an alfalfa-free casein chow (Test Diet) tominimize gastrointestinal background fluorescence. Image analysis todetermine total radiant efficiency was performed using Living Image(Caliper Life Sciences). B16F10 cells lacking tyrosinase-relatedprotein-2, B16F10-Trp2KO, were used to inoculate tumors withoutpigmentation in order to maximize the fluorescence signal-to-noise.

Fluorescently-labeled Lumican, Lumican-MSA, or MSA were intratumorallyinjected and mice were monitored for total radiant efficiency over time.The fluorescent signal from lumican (37 kDa) was retained to a greaterextent than the fluorescent signal from MSA (67 kDa) during the first 5hours post-injection (data not shown) despite the smaller molecularweight of lumican. These data suggest that the intratumoral retention oflumican is dependent, at least in part, on its collagen bindingactivity. The fluorescent signal from lumican-MSA was also retained to agreater extent than the fluorescent signal from MSA (FIG. 2A).Lumican-MSA (104 kDa) is significantly larger than lumican (37 kDa), andalthough they each possess an identical collagen binding site,lumican-MSA is retained to a greater extent than lumican alone (FIG.2A), presumably due to more rapid clearance of the smaller lumicanconstruct.

To further evaluate the effect of molecular weight on the systemicdistribution, the amount of fluorescence from serum of mice injectedwith fluorescently-labeled lumican fused to MSA over time wasdetermined. Fluorescently-labeled MSA was used as a comparator. Forassessing serum fluorescence after injection, a small volume of blood(<10 uL) was drawn by capillary action into glass micro-hematocritheparin coated tubes (VWR) from the tip of the tail at indicated timepoints. Tubes were sealed at one end with parafilm and stored uprightprotected from the light at 4 C to allow serum separation from clottedblood by gravity. Tubes were scanned using a flatbed Typhoon TrioVariable Mode Imager (GE Healthcare) (excitation laser: 633 nm; emissionfilter: 670 BP; PMT: 450-500 V) and serum fluorescence quantified usingFiji image analysis software.

As shown in FIG. 2B, the fluorescence signal from serum of mice injectedwith lumican-MSA was lower over time compared to the fluorescence signalfrom serum of mice injected with MSA as a % of injected dose. Theseresults demonstrate that lumican-MSA exhibits less systemic distributionthan MSA alone.

Lumican co-localization with collagen types I and IV was assessedfollowing intratumoral administration by immunofluorescence imaging.Briefly, B16F10 tumors were excised following intratumoral injection offluorescently-labeled lumican. The tumors were preserved overnight inperiodate-lysine-paraformaldehyde at 4° C., then cryoprotected with 30%sucrose in PBS for 8 hours at 4° C., and then frozen slowly in acryomold containing 100% OCT compound (Sakura Finetek USA Inc.) on dryice. Frozen tumor molds were sectioned to 50 micron thickness (CryStarNX70), and sections were dried at room temperature for an hour prior toantigen retrieval. For antigen retrieval, tissue sections were heated at60° C. in 10 mM sodium citrate with 0.05% Tween20 for 1 hour, washed inPBS, and then treated with 2 mg/mL of hyaluronidase for 30 minutes atroom temperature. Sections were washed in immunomix (PBS containing 0.2%wt/vol bovine serum albumin, 0.05% wt/vol sodium azide, 0.3% vol/volTriton X-10, 10% vol/vol donkey serum), then permeabilized with coldacetone for 10 minutes at −20° C., and then blocked in Immunomix for 1hour at room temperature. Staining was performed with rabbit antibodiesagainst collagen type I (ab34710, Abcam) and collagen type IV (ab6586,Abcam) at a 1:200 dilution in Immunomix overnight at room temperature ina humidifying chamber. After several PBS washes, sections were stainedwith secondary AF488-conjugated goat anti-rabbit antibody at 1:500dilution in Immunomix overnight at room temperature in a humidifyingchamber. After several washes, sections were fixed in 1% neutralbuffered formalin in PBS and mounted in VECTASHIELD anti-fade mountingmedium. Sections were imaged by confocal microscopy. By overlayingfluorescent signal of collagen type I or type IV with fluorescent signalof lumican, it was determined that lumican co-localized with bothcollagen type I and type IV in B16F10 tumors to a high extent (data notshown).

These results demonstrate that both affinity to collagen and increasedmolecular weight contribute the intratumoral retention and systemicdistribution of collagen-binding fusion proteins. These results suggestthat increasing the affinity to collagen and the molecular weight of acollagen-binding immunomodulatory molecule will increase intratumoralretention and decrease systemic distribution, thereby increasingtherapeutic effect.

Example 5: Proximity to Bound Collagen Decreases Payload Activity ofCollagen-Binding Fusion Proteins

To evaluate the effect of collagen-binding on payload activity, theintratumoral bioluminencence of Lumican-Gluc compared to ColG-Gluc wascompared in vivo. Briefly, equimolar amounts (0.3 nmol) of Lumican-Glucor GolG-Gluc were intratumorally injected into B16F10 melanomatumor-bearing mice. Less intratumoral bioluminescence was observed withLumican-Gluc compared to ColG-Gluc despite an equimolar (0.3 nmol)injection. Specifically, 4/5 mice injected with ColG-Gluc had detectablebioluminescence over background, while only 1/5 mice injected withLumican-Gluc had detectable bioluminescence over background (data notshown). Interestingly, bioluminescence signal was only detected outsidethe tumor, presumably from construct that was leaking out of the tumor.The lack of intratumoral bioluminescence from collagen-bound constructssuggests that the enzyme is not optimally functional when forced intoclose proximity to collagen. This result informs the need for a spacerprotein, like MSA, to help better separate collagen from any futurepayload.

Since collagen is an insoluble protein and thus susceptible tosolid-phase behavior like surface adsorption of proximal solubleproteins, the MSA acts as a large hydrophilic spacer protein between thepayload and lumican to protect the payload from adsorption andfunctional denatuation. For our localization approach, any solublepayload is forced into the solid-liquid interface by lumican and can beadsorbed onto a collagen fiber potentially rendering it functionallyinert (as seen with Gluc). To prevent adsorption of our payload ontocollagen, we operably link mouse serum albumin (MSA) to lumican as alarge spacer protein between the payload and collagen.

Example 6: Synergistic Effect of Immunomodulatory Collagen-BindingMolecules and Anti-Tumor Antigen Antibody in Mouse Melanoma Tumor Model

The synergy between anti-tumor antigen antibody and interleukin-2 (IL-2)is well characterized (Zhu et al., (2015) Cancer Cell 27:489-501). Theeffect of fusing IL-2 to collagen-binding molecules was evaluated bypreparing IL-2 fused to the C-terminus of Lumican-MSA. MSA wasincorporated to ensure steric access of receptors to IL-2 when bound tocollagen fibrils and also to increase the construct's molecular weightand thereby reduce diffusive flux from the tumor. As an equivalentlybioactive comparator, IL-2 was expressed as a fusion to MSA (MSA-IL2),allowing the effects of collagen binding to be largely separated fromsize-based improvements in tumor retention of small cytokines likewild-type IL-2. Thus, IL-2 was expressed as a fusion to MSA alone or asa fusion to the C-terminus of Lumican-MSA using the methods described inExample 1. His-tagged cytokine-fusion proteins were purified by TALONmetal affinity resin as described in Example 1. Cytokine-fusion proteinswere further purified by FPLC (AKTA, GE Healthcare) using a sizeexclusion chromatography column (HiLoad 16/600 Superdex 200 pg) that hadbeen pre-treated for 4 hours with 1M NaOH to remove endotoxin, thensubsequently equilibrated in sterile PBS. After purification, theproteins were buffer exchanged into sterile PBS, sterile filtered by a0.2 micron membrane filter (Pall Corporation) and confirmed to containminimal endotoxin (<0.1 EU per dose) using a chromogenic LAL assay(Lonza).

The proteins demonstrated >90% monomeric expression when evaluated byabsorbance at 280 nm during size exclusion chromatography (data notshown). Additionally, the ability of the proteins to induce cellproliferation was evaluated. CTLL-2 cells were seeded on tissue cultureplates either uncoated or coated with collagen type IV at a density of5000 cells/well. The cells were stimulated with various concentrationsof MSA-IL2, Lumican-MSA-IL2, or Lumican for 48 hours. Cell proliferationwas determined by WST-1 based colorimetric assay (Roche) according tomanufacturer's instructions. While Lumican resulted in no proliferation,treatment with MSA-IL2 and Lumican-MSA-IL2 resulted in similar levels ofcell proliferation, regardless of the presence or absence of collagentype IV (data not shown). Thus, MSA-IL2 and Lumican-MSA-IL2 haveequivalent bioactivity.

Additionally, serum levels of the IL-2 fusions following intratumoralinjection were quantified as described in Example 4.Fluorescently-labeled lumican-MSA-IL2 and MSA-IL2 were injected intoB16F10-Trp2KO tumors, and serum fluorescence levels were quantifiedfollowing injection at different time points. The fluorescence signalfrom serum of mice injected with lumican-MSA-IL2 was lower as over timeas compared to the fluorescence signal from serum of mice injected withMSA-IL2 when measured as a % of injected dose (data not shown). Theseresults demonstrate that lumican-MSA-IL2 binds to intratumoral collagenin vivo and as a result, exhibits less systemic distribution thanMSA-IL2 alone.

To characterize the anti-tumor efficacy of combinations ofimmunomodulatory collagen-binding molecules with tumor antigen-targetingantibodies, fusion proteins comprising a collagen-binding polypeptidefused to the cytokine IL-2 were assessed for synergy with a mousemonoclonal anti-TYRP1 antibody (TY99) in a B16-F10 melanoma model.

Briefly, C57BL/6 mice aged 6-8 weeks were injected subcutaneously intothe right flanks with 1×10⁶ B16-F10 mouse melanoma cells (ATCC) in 50 μLsterile PBS. Mice with established B16-F10 melanoma tumors were treatedsystemically with 100 μg/dose anti-TYRP-1 antibody (TA99) viaintraperitoneal (i.p.) injection and with 13 μg/dose of thecollagen-binding IL-2 fusion protein lumican-MSA-IL2 via intratumoralinjection (i.tu.). Mice injected with MSA-IL2 (SEQ ID NO: 121) (9μg/dose) or lumican (SEQ ID NO: 182) (4 μg/dose) were used ascomparators. Mice injected with IL-2 fusion proteins (lumican-MSA-IL2(SEQ ID NO: 120) or MSA-IL2) received the equivalent of 0.11 nmol/doseof IL-2. Animals were euthanized at a euthanasia endpoint, which waseither 20% total body weight loss or tumor area exceeding 100 mm²(length×width).

Percent survival of mice with tumors treated with MSA-IL2,lumican-MSA-IL2, lumican alone or in combination with TA99 is shown inFIG. 3A-3B. Monotherapy of mice administered lumican-MSA-IL2 or MSA-IL2alone imparted a limited survival benefit (FIG. 3A). The combination ofTA99 and lumican provided no survival benefit (FIG. 3B), however,administration of a combination of TA99 and either MSA-IL-2 orlumican-MSA-IL2 showed a synergistic survival benefit to mice (FIG. 3B).The combination with lumican-MSA-IL2 imparted a greater survival benefitcompared to the combination with MSA-IL2 (FIG. 3B).

After cessation of treatment, several mice developed localized skindepigmentation or vitiligo which is indicative of a melanocyte-specificT cell response. Nearly all mice injected with lumican-MSA-IL2 and TA99developed a patch of vitiligo localized to the site of injection (16 outof 17 mice) whereas only one out of 17 mice treated with MSA-IL2 andTA99 displayed this side effect. Together, these observations indicatethat lumincan-MSA-IL2 anchors IL-2 intratumorally and enhances IL-2'ssynergy with TA99 by increasing anti-tumor T cell responses and overallsurvival.

To determine if intratumoral injection of lumican-MSA-IL-2 was necessaryfor the improved outcomes when used in combination with TA99, weevaluated the efficacy of this combination when lumican-MSA-IL2 wasinjected into other corporeal sites. When lumican-MSA-IL2 wasadministered peritumorally (peri.tu) (i.e. adjacent to the lesion) (FIG.3C) or intranodally (i.e. into the tumor draining inguinal lymph node)(FIG. 3D), efficacy was attenuated. All survival benefit of thecombination diminished when lumican-MSA-IL2 was administeredsubcutaneously at the tail base, 2 cm distal to the tumor site (FIG.3C).

These results show that treatment of tumor-bearing mice withlumican-MSA-IL2 in combination with TA99 provides a synergisticanti-tumor effect and that intratumoral localization of IL-2 is requiredfor maximal efficacy of this combination therapy.

Example 7: Synergistic Effect of Immunomodulatory Collagen-BindingMolecules and Anti-Tumor Antigen Antibody is Dependent on CD8+ T Cells,Dendritic Cells, and IFNγ

High dose IL-2 supports the proliferation and effector functions of Tcells and NK cells, but also promotes neutrophilia and eosinophilia(Macdonald et al., (1990) Br J Haematol 76(2):168-173; Li et al., (1996)Inflammation 20(4):361-372). Given the known diverse effects of IL-2 onimmune cells, the contribution of distinct leukocytes types to thetherapeutic efficacy of lumican-MSA-IL2 was determined byantibody-mediated cellular depletions. Immune cell subsets or IFNγ weredepleted by intraperitoneal (i.p.) administration of depleting antibodybeginning one day before the first treatment until one week after thelast treatment. TA99 and lumican-MSA-IL2 were administered as describedfor FIG. 3B. CD8+ T cells, NK cells or neutrophils were depleted using400 μg of anti-CD8α (2.43, BioXCell), anti-NK1.1 (PK136, BioXCell), oranti-Ly6G (1A8, BioXCell) antibody every four days, respectively.Macrophages or soluble IFNγ were depleted using 300 μg of anti-CSF1R(AFS98, BioXCell) or 200 μg of anti-IFNγ (XMG1.2, BioXCell) every otherday, respectively. Eosinophils were depleted using 1 mg of anti-IL-5(TRFK5, BioXCell).

To assess contribution of immune cell subsets, 6-8 week old C57BL/6wildtype (WT) or BatF3^(−/−) mice (B6.129S(C)-Batf3^(tm1Kmm)/J; JacksonLaboratory), which are deficient in cross-presenting dendritic cells(DCs), were injected subcutaneously into the right flanks with 1×10⁶B16-F10 mouse melanoma cells (ATCC) in 50 μL sterile PBS. Depletion ofnatural killer (NK) in WT mice did not alter efficacy of lumican-MSA-IL2in combination with TA99 (FIG. 4). Depletion of neutrophils,eosinophils, or macrophages in wildtype mice also did not alter efficacyof lumican-MSA-IL2 in combination with TA99 (data not shown), indicatingthat no single innate cell population was solely responsible for tumorcontrol. However, depletion of CD8+ T cells (anti-CD8a),cross-presenting DCs (BatF3^(−/−)), and IFNγ (anti-IFNγ) did alterefficacy of treatment indicating that they were indispensable for tumorrejection (FIG. 4). Survival statistics were determined by log-rankMantel-Cox test.

These results show that treatment of tumor-bearing mice withlumican-MSA-IL2 in combination with TA99 provides a synergisticanti-tumor effect that is dependent on CD8+ T cells, dendritic cells andIFNγ.

Example 8: Combination of Immunomodulatory Collagen-Binding Moleculesand Anti-Tumor Antigen Antibody Establishes Protective Memory andInduces Systemic Tumor-Specific Cellular Immunity

The durable disease-free survival and the reliance on components ofadaptive immunity observed after treatment with lumican-MSA-IL2 incombination with TA99 as shown above suggested that cured tumor-freemice might be resistant to tumor re-challenge. To determine if curedtumor-free mice treated with lumican-MSA-IL2 in combination with TA99,cured C57BL/6 mice from the experiment shown in FIG. 3B werere-challenged on day 100 with 1×10⁶ B16-F10 cells inoculated on thecontralateral flank. A majority of long-term survivors treated withlumican-MSA-IL2 in combination with TA99 rejected a rechallenge withB16-F10 inoculated on the contralateral flank (9/15 mice).

Although tumor eradication by cell-mediated immunity requires systemicimmune activation (Spitzer et al., (2017) Cell 168:487-502), thestrictly localized patch of vitiligo observed following treatment withlumican-MSA-IL2 in combination with TA99 (see Example 6) prompted anevaluation of tumor-specific T cell responses outside the treated tumorlesion. To determine if treatment with lumican-MSA-IL2 in combinationwith TA99 induces a systemic anti-tumor T cell response, an IFNγ ELISPOTwas performed using splenocytes harvested four days after treatment ofmice as described in FIG. 3B. Treatment with TA99 in combination withMSA-IL2 or lumican were used as comparators. The number of IFNγ spotforming units (SFUs) in response to stimulation with B16F10 target cellswas quantified. Treatment with TA99 in combination with lumican-MSA-IL2yielded more peripheral splenocytes expressing IFNγ than in combinationwith MSA-IL2. Specifically, treatment with TA99+Lumican-MSA-IL2 resultedin approximately 20 SFUs per 1 million splenocytes, while treatment withTA99+MSA-IL2, with TA99+lumican, or no treatment resulted in less thanapproximately 7 SFUs per 1 million splenocytes.

To confirm the intracellular IFNγ staining on splenocytes was generatedby tumor-specific CD8+ T cells, splenocytes were harvested four daysafter treatment of mice as described in FIG. 3B. Harvested splenocyteswere simulated with irradiated B16-F10 or 4T1 for 12 hours in thepresence of brefeldin A and subsequently stained for surface markers(CD4, CD3 and CD8) and intracellular IFNγ (n=5 mice/group). FIG. 5Ashows the quantification of IFNγ+cells among live CD45+CD3+CD8+ T cellsas determined by flow cytometry. Data were analyzed by one-way ANOVAwith Tukey's multiple comparison test.

The results shown in FIG. 5A demonstrate that stimulation withirradiated B16-F10 tumor cells, but not 4T1 tumor cells, induced theexpression of IFNγ in splenocytes, confirming that peripheralB16F10-specific CD8+ T cell responses in spleen were induced bytreatment of tumor-bearing mice with lumican-MSA-IL2 in combination withTA99. These results also show that treatment of tumor-bearing mice withTA99 in combination with tumor collagen-anchored lumican-MSA-IL2 induceda greater tumor-specific systemic response than in combination withunanchored MSA-IL2.

To determine the ability of peripheral effectors (e.g., peripheraltumor-specific T cells) induced by treatment with TA99 in combinationwith lumican-MSA-IL2 to control a distant untreated tumor lesion whereexogenous cytokine support is limited, mice were inoculated 1×10⁶B16-F10 cells subcutaneously to establish tumors on both flanks andadministered TA99 systemically (i.p) and IL-2 (as lumican-MSA-IL2 orMSA-IL2) intratumorally (i.tu) into only the right, or ipsilateral,tumor. FIG. 5B shows the mean tumor areas for uninjected contralateraltumors and for intratumorally-injected ipsilateral tumors over time. Theresults shown in FIG. 5B demonstrate that the combination of TA99 withMSA-IL2, which can leak out of the ipsilateral tumor and to thecontralateral lesion after injection, imparted some contralateral tumorcontrol but most mice succumbed to tumor burden. In contrast, thecombination of TA99 with lumican-MSA-IL2, which is isolated to theipsilateral tumor, halted both ipsilateral and contralateral tumorgrowth leading to durable cures in several mice. These results showsystemic anti-tumor response elicited by anchoring IL-2 via tumorcollagen-binding is superior to diffuse IL-2 stimulation in controllingand eradicating disseminated disease.

Example 9: Treatment of Tumor-Bearing Mice with a Collagen-Binding IL-12Fusion Protein does not Induce IL-12-Related Weight Loss

After observing improvement of anti-tumor therapeutic effects bycollagen-anchoring of IL-2 as shown in the Examples above, the effect(s)of anchoring IL-12, another dose-limited cytokine, to collagen wasevaluated. IL-12 acts as a key regulator in type-1 cell mediatedimmunity, a pathway known to be critical for effective anti-tumorresponses (Green et al., (2017) J Biol Chem 292:13925-13933). Despitepromising preclinical work, severe toxicities and fatalities halted anearly clinical trial administering systemic IL-12 (Lasek et al., (2014)Cancer Immunol Immunother 63(5):419-435). IFNγ, induced by IL-12stimulation of NK cells and T cells, was implicated in the toxicity,however, IL-12 is also inextricably coupled to its efficacy (Leonard etal., (1997) Blood 90:2541-2548).

Thus, IL-12 was expressed and purified as a fusion to MSA alone or as afusion to the N-terminus of Lumican-MSA (IL12-MSA-Lumican) using themethods described in Examples 1 and 6. Briefly, murine IL-12 wasexpressed in a single chain format with a 15 amino acid glycine-serinelinker between the p40 and p35 subunits (scIL12). To generate acollagen-anchoring version of IL-12, scIL12 was fused to the N-terminusof lumican with an MSA spacer, henceforth referred to asIL12-MSA-Lumican (SEQ ID NO: 123). IL12-MSA (SEQ ID NO: 122), anon-anchoring version of IL-12, was used as a comparator. The IL12-MSAand IL12-MSA-Lumican proteins demonstrated >90% monomeric expressionwhen evaluated by absorbance at 280 nm during size exclusionchromatography (data not shown).

Additionally, serum levels of the IL-12 fusions following intratumoralinjection were quantified as described in Example 4.Fluorescently-labeled IL12-MSA-lumican and IL12-MSA were injected intoB16F10-Trp2KO tumors, and serum fluorescence levels were quantifiedfollowing injection at different time points. The fluorescence signalfrom serum of mice injected with IL12-MSA-lumican was lower as over timeas compared to the fluorescence signal from serum of mice injected withIL12-MSA when measured as a % of injected dose (data not shown). Theseresults demonstrate that IL12-MSA-lumican binds to intratumoral collagenin vivo and as a result, exhibits less systemic distribution thanIL12-MSA alone.

To determine if retaining IL-12 to the tumor via collagen-binding wouldameliorate IL-12-mediated toxicities, IL-12 fused to lumican-MSA wasevaluated in a mouse melanoma tumor model. C57BL/6 mice, aged 6-8 weeks,were inoculated with 1×10⁶ B16-F10 melanoma cells on day 0. Weightchange from baseline of these mice was monitored after treatment witheither intratumoral (i.tu.) injections of PBS (n=6), 17.8 μg/doseIL12-MSA (n=7), or 23.1 μg/dose IL12-MSA-Lumican (n=7), orintraperitoneal (i.p.) injection of 17.8 μg/dose IL12-MSA (n=7) on day 6and day 12. Mice injected with IL-12 fusion proteins (IL12-MSA-lumicanor IL12-MSA) received the equivalent of 140 pmol/dose of IL-12.

As shown in FIG. 6A, intratumoral administration of IL12-MSA in B16-F10tumor-bearing mice lead to significant weight loss, a noninvasivereadout for systemic cytokine (e.g., IFNγ) toxicity. Systemicadministration of IL12-MSA by intraperitoneal injection resulted in anidentical weight loss profile. In contrast, an equimolar intratumoralinjection of IL12-MSA-Lumican did not cause weight loss. Collectively,these results demonstrate that local administration in the absence of aneffort towards intratumoral retention via collagen binding isinsufficient in alleviating IL-12-mediated toxicity and thatcollagen-anchoring provided sufficient intratumoral confinement to curbovert systemic toxicities of IL-12, resulting in improved therapeuticindex.

The survival of animals treated as in FIG. 6A was also evaluated. Micewere euthanized according to the criteria described in Example 6. Asshown in FIG. 6B, treatment with an IL12 fusion improved survival overuntreated control mice or mice treated with lumican. Treatment withIL12-MSA-Lumican resulted in a modest improvement in survival comparedto IL12-MSA.

The dose of IL12 used to treat B16F10 tumors was titrated to determinedose-dependent effects on toxicity and anti-tumor efficacy. Miceinoculated with B16F10 tumors on day 0 as described above were treatedon day 5 with an intratumoral injection of IL12-MSA or IL12-MSA-Lumicanat different doses. Untreated control mice received an intratumoralinjection of PBS. The effect of dose was evaluated on tumor area (e.g.,measure of efficacy) and % weight change (e.g., measure of toxicity).The doses of IL12-MSA and IL12-MSA-Lumican that were evaluated includedthe mass equivalent of 140 pmol IL12, 14 pmol IL12, 1.4 pmol IL12, or0.14 pmol IL12. IL12-MSA-Lumican showed no toxicity at any dose tested,but demonstrated reduced efficacy at a 1.4 or 0.14 pmol dose of IL12(data not shown). While IL12-MSA demonstrated both reduced toxicity andreduced efficacy with decreased dose (data not shown). Thus to evaluateIL12 fusions in combination with other therapeutic agents, a 14 pmolIL12 dose was identified as having a tolerable toxicity index whilemaintaining therapeutic efficacy.

Example 10: Synergistic Anti-Tumor Effect of Collagen-Binding IL-2 andIL-12 Fusion Proteins in a Mouse Tumor Model

Combinations to potentiate the anti-tumor effects of IL-12 have beentheorized but safety concerns have largely precluded their actualization(Lasek et al., (2014) Cancer Immunol Immunother 63(5):419-435). Theimproved therapeutic index of IL12-MSA-lumican, as indicated by theabsence of treatment-related weight loss in Example 9, prompted theevaluation of this cytokine in combination with other therapeuticagents. IL-2 and IL-12 are known to engage complementary signalingpathways to stimulate NK cells and T cells (Wigginton & Wiltrout (2002)Expert Opin Biol Ther 2:513-524) Additionally, IL-2 upregulates theexpression of a IL-12 receptor subunit beta 2 (Wang et al., (2000) Blood95:3183) and IL-12 sustains surface expression the high-affinity IL-2receptor CD25 (Starbeck-Miller et al., (2013) J Exp Med 211:105-120). Byreciprocal positive feedback, IL-2 and IL-12 augment and prolong theeffect of each other (Wigginton et al., (1996) J Natl Cancer Inst88:38-43). Despite promising efficacy, several clinical trials aroundthis combination have been terminated (Wigginton & Wiltrout (2002)Expert Opin Biol Ther 2:513-524; Gollob et al., (2003) J Clin Oncol21:2564-2573; Addison et al., (1998) Gene Ther 5:1400-1409; Wigginton etal., (2001) J Immunol 166:1156-1168). Notably, the combination of IL-2and IL-12 also significantly enhances the production of IFN-γ by T cellsand NK cells (Wigginton & Wiltrout (2002) Expert Opin Biol Ther2:513-524).

To evaluate the therapeutic effect(s) of a combination ofimmunomodulatory collagen-binding molecules comprising IL-2 or IL-12,lumican-MSA-IL2 and IL12-MSA-lumican were tested in combination in amouse melanoma tumor model essentially as described in Example 9.However, given the known toxicities arising from administration ofnon-collagen-anchored cytokines in combination described above, thedosages of IL12-MSA-lumican and IL12-MSA-IL-12 were reduced to 1/10 ofwhat was previously administered in Example 9 to 14 pmol/dose. As inExample 6, mice administered lumican-MSA-IL2 received 13 μg/dose. Miceadministered MSA-IL2 received 9 μg/dose. Mice injected with IL-2 fusionproteins (lumican-MSA-IL2 or MSA-IL2) received the equivalent of 0.11nmol/dose of IL-2.

Intratumoral administration of the reduced dose (14 pmol/dose) ofIL12-MSA alone or MSA-IL2 alone did not result in weight loss orsignificant tumor growth delay in B16-F10 tumor-bearing mice (data notshown). In contrast, the administration of IL12-MSA in combination withMSA-IL2 resulted in weight loss and increased survival (FIG. 7). Incontrast, combination treatment using the collagen-anchoring versions,IL12-MSA-Lumican and Lumican-MSA-IL2, increased survival to a greaterextent than combination of IL12-MSA and MSA-IL2 and did not result inconcomitant weight loss (FIG. 7).

These results demonstrate that treatment of tumor-bearing mice with acombination of collagen-binding IL12-MSA-lumican and lumican-MSA-IL2resulted in increased survival of mice to a greater extent thantreatment with a combination of non-collagen-binding IL12-MSA andMSA-IL2. Furthermore, these results show that treatment of mice with acombination of collagen-binding IL12-MSA-lumican and lumican-MSA-IL2prevented treatment-related toxicity associated with co-administrationof IL-12 and IL-2, thereby providing a therapeutic modality for thiscytokine combination.

Example 11: Synergistic Effect of Collagen-Binding IL-2 and IL-12 FusionProteins is Dependent on CD8+ T Cells and Dendritic Cells

The immune cell types responsible for the efficacy of intratumoralIL12-MSA-Lumican and Lumican-MSA-IL2 combination therapy was determinedby antibody-mediated cellular depletions essentially as described inExample 7. As shown in FIG. 8A, CD8+ T cells and cross-presentingdendritic cells are indispensable for efficacy, as depletion of thesecell types reduces survival of tumor-bearing mice treated intratumorallywith a combination of IL12-MSA-Lumican and Lumican-MSA-IL2. In contrast,depletion of NK cells, neutrophils, eosinophils, or macrophages did notsignificantly affect the survival outcome (FIG. 8B). Antibody-mediateddepletion of IFNγ, a cytokine known to be amplified by concurrent IL-2and IL-12 stimulation (Gollob et al., (1999) J Immunol162(8):4472-4481), also did not significantly alter survival (FIG. 8A),however, the lack of effect might be attributed to insufficientdepletion of IFNγ.

To further evaluate the contribution of immune cell types toward theanti-tumor efficacy provided by the combination of IL12-MSA-lumican andlumican-MSA-IL2 in tumor-bearing mice, immunophenotyping oftumor-infiltrating immune cells was performed. Mice were inoculated with1 million B16F10 tumor cells on day 0 and treated with eitherintratumoral injection of PBS, IL12-MSA and MSA-IL2, or IL12-MSA-Lumicanand Lumican-MSA-IL2 on day 5. Tumors were excised on day 11. Immune cellinfiltrates of tumors were analyzed as previously described. (Moynihanet al., (2016) Nat Med 22:1402-1410); Zhu et al., (2015) Cancer Cell27:489-501). Briefly, resected tumors were weighed, dissociated intosmall pieces, incubated in RPMI-1640 containing 1 mg/mL collagenase anddispase (Roche) and 25 ug/mL DNase I (Roche) for 30 minutes at 37 C.Further mechanical dissociation was used to generate a single cellsuspension for staining. Cells were analyzed on BD FACS LSRFortessa™ anddata was analyzed using FlowJo® (FlowJo, Inc). Cells were stained forsurface and intracellular markers to delineate cell types as follows:

NK cells (live CD45⁺ CD3⁻ NK1.1⁺)

Treg cells (live CD45⁺ CD3⁺ NK1.1⁻ CD4⁺ CD8⁻ CD25⁺ FoxP3⁺)

CD4 cells (live CD45⁺ CD3⁺ NK1.1⁻ CD4⁺ CD8⁻ CD25^(+/−) FoxP3⁻)

CD8 T cells (live CD45⁺ CD3⁺ NK1.1⁻ CD4⁻ CD8⁺)

Monocytes/Macrophages (live CD45⁺ CD11b⁺ Ly6G⁻ CD11c⁻ F4/80⁺)

CD11b⁺ DC (live CD45⁺ CD11b⁺ MHCII⁺ CD11c⁺)

CD11b⁻ DC (live CD45⁺ CD11b⁻ MHCII⁺ CD11c⁺)

Neutrophil (live CD45⁺ CD11b⁺ Ly6G⁺)

As shown in FIG. 8C, the fold change of CD8+ T cells in tumorinfiltrates from mice treated with a combination of IL12-MSA-Lumican andLumican-MSA-IL2 (Lumican versions) relative to treatment with PBS ishigher than the fold change from mice treated with IL12-MSA+MSA-IL2 (MSAversions) relative to treatment with PBS. Furthermore, tumors treatedwith the lumican-MSA cytokine fusions had more infiltrating CD8+ T cellscompared to tumors treated with the MSA cytokines fusions six days afterinitial treatment (FIG. 8D) and had higher surface expression of PD-1(FIG. 8E).

The production of tumor-specific T cells in response to treatment wasfurther evaluated in splenocytes isolated at six days followingtreatment by IFNγ ELISPOT. The number of IFNγ spot-forming units (SFU)in response to stimulation with B16F10 target cells was quantified.Treatment with lumican-MSA cytokine fusions yielded approximately 20 SFUper 1 million splenocytes, compared to approximately 15 SFU per 1million splenocytes for MSA cytokine fusions or approximately 2 SFU per1 million splenocytes for untreated animals. Thus, treatment withlumican-MSA cytokine fusions resulted in an increase in the number ofperipheral tumor-specific T cells compared to no treatment or treatmentwith MSA-cytokine fusions.

These results demonstrate that the anti-tumor efficacy (e.g., increasedsurvival) provided by the combination of IL12-MSA-Lumican andLumican-MSA-IL2 is dependent on CD8+ T cells and dendritic cells.Furthermore, these results demonstrate that intratumoral treatment oftumor-bearing mice with a combination of IL12-MSA-Lumican andLumican-MSA-IL2 provides anti-tumor efficacy, at least in part, byinducing the infiltration of activated CD8+ T cells into the tumor andinducing production of peripheral tumor-specific T cells.

Example 12: Synergistic Effect of Collagen-Binding IL-2 and IL-12 FusionProteins and Anti-PD-1 Antibody Combination in a Mouse Melanoma TumorModel

The upregulation of surface PD-1 on tumor infiltrating CD8 T cells inresponse to lumican-cytokine treatment as described in Example 11prompted an evaluation of the anti-tumor efficacy of a combination of ananti-PD-1 antibody (clone 29F. 1A12, BioXCell), IL12-MSA-lumican, andlumican-MSA-IL2 in a mouse melanoma tumor model. Briefly, C57BL/6 miceaged 6-8 weeks were inoculated with 1×10⁶ B16-F10 tumor cellssubcutaneously into the right flank on day 0. Tumor-bearing mice weretreated with a combination of Lumican-MSA-IL2 and IL12-MSA-Lumican or acombination of MSA-IL2 and IL12-MSA intratumorally at dosages asdescribed in Example 10 and with anti-PD-1 antibody (200 μg/dose)intraperitoneally on day 5 and day 11. Percent weight change frombaseline and percent survival was monitored and is shown in FIG. 9.

As shown in FIG. 9, the inclusion of anti-PD-1 antibody in combinationwith IL12-MSA and MSA-IL2 or with IL12-MSA-Lumican and Lumican-MSA-IL2did not alter the weight loss trends observed previously (FIG. 7).However, the addition of anti-PD-1 antibody improved survival outcomesfor mice treated with the IL12-MSA+MSA-IL2 combination but did notfurther improve survival for mice treated with theIL12-MSA-Lumican+Lumican-MSA-IL2 combination.

Localized vitiligo occurred more frequently in mice treated withanti-PD-1 antibody, IL12-MSA-Lumican, and Lumican-MSA-IL2 (4/5 mice withvitiligo at injection site) compared to mice treated with anti-PD-1antibody, IL12-MSA, and MSA-IL2 (1/5 mice with vitiligo at injectionsite). Furthermore, more survivors from cytokine (IL12-MSA+MSA-IL2 orIL12-MSA-Lumican+Lumican-MSA-IL2) and anti-PD-1 treatment compared tosurvivors from IL12-MSA-Lumican+Lumican-MSA-IL2 treatment in the absenceof anti-PD-1 treatment were protected against a subsequent tumorre-challenge with B16-F10 tumor cells. While 4/4 mice treated withLumican-MSA cytokines+anti-PD-1 and 2/2 mice treated with MSAcytokines+anti-PD-1 rejected re-challenge tumors, only 1/3 mice treatedwith Lumican-MSA cytokines only rejected re-challenge.

These results demonstrate treatment of tumor-bearing mice with acombination of anti-PD-1, IL12-MSA-Lumican, and Lumican-MSA-IL2increased the occurrence of treatment-induced vitiligo and effectiveimmunological memory thus demonstrating a synergistic effect inenhancing T cell responses generated from localized IL-2 and IL-12treatment.

Example 13: Synergistic Effect of Collagen-Binding IL-2 and IL-12 FusionProteins and Anti-PD-1 Antibody Combination in Mouse Mammary and ColonCarcinoma Tumor Models

The anti-tumor effect(s) of a combinations of collagen-binding IL-2 andIL-12 fusion proteins with an anti-PD-1 antibody were further assessedin an EMT6 mammary carcinoma model. Briefly, 1×10⁶ EMT6 mouse mammarycarcinoma cells (ATCC) or 1×10⁶ MC38 mouse colon carcinoma cells(National Cancer Institute, Bethesda, Md.) were resuspended in 50 μL ofsterile PBS were injected subcutaneously into the right flanks ofC57BL/6 female mice on day 0. EMT6 tumor-bearing mice were treated withLumican-MSA-IL2+IL12-MSA-Lumican (i.tu.), anti-PD-1 antibody alone(i.p., 200 μg/dose) or anti-PD-1 antibody in combination withLumican-MSA-IL2 and IL12-MSA-Lumican (i.tu) at dosages as described inExample 10 on day 5, day 11 and day 17. MC38 tumor-bearing mice weretreated with a combination of Lumican-MSA-IL2+IL12-MSA-Lumican (i.tu.),anti-PD-1 antibody alone (i.p., 200 μg/dose) or in combination withLumican-MSA-IL2+IL12-MSA-Lumican (i.tu.) at dosages as described inExample 10 on day 5, day 11 and day 17. For each tumor model, tumor area(mm²) and percent survival was monitored over time and is shown in FIG.10A-10B. Survival statistics determined by log-rank Mantel-Cox test.

As shown in FIG. 10A, treatment of mice bearing EMT6 mammary carcinomatumors with an anti-PD-1 antibody in combination with Lumican-MSA-IL2and IL12-MSA-Lumican resulted in resolution of tumor lesions (asindicated by absence of measurable tumor area) and increase survival toa greater extent than treatment of tumor-bearing mice with anti-PD-1antibody alone.

As shown in FIG. 10B, treatment of mice bearing MC38 colon carcinomatumors with an anti-PD-1 antibody in combination with Lumican-MSA-IL2and IL12-MSA-Lumican resulted in a decrease in tumor area and increasedsurvival to a greater extent than treatment of tumor-bearing mice withanti-PD-1 antibody alone or with the IL12-MSA-Lumican+Lumican-MSA-IL2combination alone.

These results show that treatment of tumor-bearing mice with ananti-PD-1 antibody in combination with IL12-MSA-Lumican+Lumican-MSA-IL2results in a synergistic anti-tumor effect in both EMT6 mammarycarcinoma and MC38 colon carcinoma models.

Example 14: Synergistic Effect of Collagen-Binding IL-12 Fusion Proteinand Cancer Vaccine in a Mouse Melanoma Tumor Model

To further evaluate the ability of a collagen-binding IL-12 fusionprotein to synergistically potentiate anti-tumor treatments, theanti-tumor effect(s) of the collagen-binding IL-12 fusion proteinIL12-MSA-Lumican in combination with a cancer vaccine was assessed in anB16-F10 mouse melanoma model. Briefly, 1×10⁶ B16-F10 cells (ATCC),resuspended in 50 μL of sterile PBS, were inoculated subcutaneously intothe right flank of 6-8 week old C57BL/6 female mice on day 0. A cancervaccine, comprising 90 μg of a lymph node-targeting moiety fused topeptides derived from B16-F10-associated antigens TYRP-1 and a modifiedgp100 peptide (EGP) and 50 g of cyclic dinucleotides adjuvants(Invivogen), was administered subcutaneously at the base of the tailwith a prime dose on day 5 and boost dose on day 11 and day 17.Subcutaneous administration of the cyclic dinucleotide adjuvant and thecancer vaccine was evaluated for priming of an antigen-specific CD8 Tcell response. Briefly, peripheral blood was collected on day 16 andstimulated for 6 hours with peptide antigens Trp1 and EGP. Brefeldin Awas included for the final 4 hours of incubation. Peripheral blood cellswere subsequently stained for surface markers and intracellular IFNγ andanalyzed by flow cytometry. The percentage of IFNγ⁺ cells among liveCD45⁺ CD3⁺ CD8⁺ T cells was evaluated. Vaccination alone or incombination with IL12-MSA-Lumican improved priming of antigen-specificCD8 T cells (data not shown).

Weight change from baseline of each mouse, tumor area, and survival ofmice treated with intratumoral (i.tu.) injections of PBS (n=12) or IL-12(n=10 for IL12-MSA; n=10 for IL12-MSA-Lumican), or vaccine (n=7) alone,or vaccine and IL12 (n=7 for IL12-MSA; n=7 for IL12-MSA-Lumican) on days5, 11, and 17 were monitored over time (FIG. 11, left to right).

As shown in FIG. 11, vaccination alone did not affect weight relative tomice treated with PBS (left panel), modestly delayed B16F10 tumor growth(middle panel), and modestly increased survival of mice (right panel).In contrast, co-administration of the cancer vaccine with IL-12, usingeither IL12-MSA or IL12-MSA-Lumican, resulted in a synergistic reductionin tumor growth (middle panel) and increase in survival (right panel).The cancer vaccine administered in combination with IL12-MSA-Lumicanextended survival longer than combination with IL12-MSA. Additionally,the vaccine with IL12-MSA lead to treatment-induced weight loss that wasundetected with IL12-MSA-Lumican (right panel).

These results demonstrate that administration of a cancer vaccine incombination with collagen anchored IL-12 (IL12-MSA-lumican) totumor-bearing mice results in a synergistic anti-tumor effect,decreasing tumor growth and increasing percent survival to a greaterextent that administration of the cancer vaccine or IL12-MSA-lumicanalone. These results show that intratumoral collagen-anchoring of acytokine (e.g., IL-12) synergistically improves the tumor control of acancer vaccine.

Example 15: Synergistic Effect of Collagen-Binding IL12 Fusion Proteinand CAR-T Cells in a Mouse Melanoma Tumor Model

To further evaluate the ability of a collagen-binding IL-12 fusionprotein to synergistically potentiate anti-tumor treatments, theanti-tumor effect(s) of the collagen-binding IL-12 fusion proteinIL12-MSA-Lumican in combination with CAR-T cells was assessed in anB16-F10 mouse melanoma model. Briefly, 0.5×10⁶ B16-F10 cells (ATCC),resuspended in 50 μL of sterile PBS, were inoculated subcutaneously intothe right flank of 6-8 week old C57BL/6 female mice on day 0.B16F10-specific CAR-T cells were generated by transducing CD3+splenocytes to express a CAR composed of single-chain variable fragment(scFv) of TA99 fused to costimulatory CD28 and CD3 ζ signaling domains.To ensure CAR-T cell engraftment, all mice were preconditioned withtotal body irradiation the day prior to a bolus injection of 10 millionCAR-T cells intravenously (i.v.). Weight change from baseline, tumorarea, and survival of mice treated with intratumoral (i.tu.) injectionsof PBS (n=9) or IL-12 (n=6 for IL12-MSA; n=5 for IL12-MSA-Lumican), orCAR-T (n=12) alone, or CAR-T and IL12 (n=7 for IL12-MSA; n=5 forIL12-MSA-Lumican) on days 5 and 11 were monitored over time (FIG. 12,left to right).

As shown in FIG. 12, treatment with CAR-T cells or IL12-MSA-Lumican (2.3ug/dose) alone decreased tumor area (middle panel) and increasedsurvival (right panel). However, administration of a combination ofCAR-T cells and IL12-MSA-Lumican to tumor-bearing mice resulted indurable tumor regression, reducing tumor area and increasing survival toa greater extent that treatment with either CAR-T cells orIL12-MSA-Lumican alone. Treatment with CAR-T cells in combination withIL12-MSA also resulted in tumor regression and modest improvement insurvival. However, the combination treatment demonstrated significanttoxicity as shown by the large reduction in animal body weight lossfollowing treatment. Such toxicity was not observed for the combinationof CAR-T cells and IL-12-MSA-lumican.

These results demonstrate that administration of tumor-antigen specificCAR-T cells in combination with collagen anchored IL-12(IL12-MSA-lumican) to tumor-bearing mice results in a synergisticanti-tumor effect, decreasing tumor growth and increasing percentsurvival to a greater extent that administration of the CAR-T cells orIL12-MSA-lumican alone. These results show that intratumoralcollagen-anchoring of a cytokine (e.g., IL-12) synergistically improvesthe tumor control of tumor-antigen specific CAR-T cells.

Example 16: Neoadjuvant Administration of a Collagen-Binding IL-12Fusion Protein in Combination with PD-1 Checkpoint Blockade PreventsMetastatic Recurrence in a Mouse Breast Tumor Resection Model

To further evaluate the ability of a collagen-binding IL-12 fusionprotein to synergistically potentiate anti-tumor treatments, theanti-tumor effect(s) of the collagen-binding IL-12 fusion proteinIL12-MSA-Lumican in combination with an anti-PD-1 antibody (clone29F.1A12, BioXCell) was assessed in an 4T1 mouse breast tumor resectionmodel and compared to scIL12 combined with anti-PD-1. Briefly, 0.5×10⁶luciferase-expressing 4T1-Luc cells (MIT, Cambridge, Mass.), resuspendedin 100 μL of sterile PBS, were injected into the mammary fat pad of 6-8week old BALB/c female mice on day 0. Prior to surgical resection ofprimary lesions, mice were treated with IL12-MSA-Lumican or scIL12intratumorally and anti-PD-1 systemically (neoadjuvant therapy).Neoadjuvant therapy (anti-PD-1, 200 μg/dose, administered i.p.+IL12-MSA-lumican, 4.6 μg/dose (30 pmol IL12/dose) or scIL12, 1.9μg/dose (30 pmol IL12/dose)), administered i.tu.) was administered onday 7 and 13 and primary tumors were surgically excised on day 16.Post-operation mice were monitored by in vivo imaging (IVIS) formetastases. FIG. 13 shows total body weight change during neoadjuvanttreatment (left panel), primary tumor growth and weight (middle panel)and percent survival (right panel) of mice treated with intratumoral(i.tu.) injections of IL-12 (n=5 for scIL12; n=5 for IL12-MSA-Lumican)and intraperitoneal (i.p.) injection of anti-PD-1 on day 7 and 13.Arrows indicate time of treatment and cross indicates time of surgery.

As shown in FIG. 13, the combination of either version of IL-12 (scIL12or IL12-MSA-Lumican) and anti-PD-1 antibody was not overtly toxic basedon the absence of weight loss (left panel). However, the neoadjuvanttherapy with IL12-MSA-Lumican led to more primary tumor shrinkage thanthe non-anchoring version scIL-12 (middle panel). After surgery, micewere monitored for metastases by in vivo bioluminescence imaging.IL12-MSA-Lumican completely protected mice from metastatic growth whileseveral mice treated with scIL-12 relapsed.

These results demonstrate that administration of anti-PD-1 antibody incombination with collagen anchored IL-12 (IL12-MSA-lumican) totumor-bearing mice results in a synergistic anti-tumor effect,decreasing primary tumor growth and increasing percent survival aftersurgical resection of tumor to a greater extent that administration ofthe a combination of non-collagen-binding IL-12 (scIL12) and anti-PD-1antibody. These results demonstrate that collagen-anchoring IL-12 in aneoadjuvant setting improves postoperative outcomes.

Example 17: Collagen-Binding Chemokine Fusion Proteins Induce ImmuneCell Migration and Tumor Infiltration

T cell infiltration is critical for durable anti-tumor immunity. Acorrelation exists between T-cell infiltration and tumor-cell derivedexpression of CCL3, CCL4, and CCL5. Spranger et al., (2015) Nature523:231-235 (2015); Spranger et al. (2016) Proc Natl Acad Sci USA 113,E7759-E7768 (2016). CCL3 (MIP-1α) binds with high affinity to CCR1, andlow affinity to CCR5 thereby mediating the recruitment of T cells, Bcells and monocytes. CCL4 (MIP-1b) binds to CCR5 mediating generallymphocyte recruitment. CCL5 (RANTES) binds several chemokine receptors(CCR1, CCR3, CCR4, CCR5) and thereby attracts monocytes, T cells,eosinophils, and other immune cells. T-cell recruiting chemokines arealso more prevalent in tumors undergoing productive immune-mediatedregression. Liang et al., (2016) Proc Natl Acad Sci USA 113:5000-5005;Schlecker et al., (2012) J Immunol 189:5602-5611; Brewitz et al., (2017)Immunity 46:205-219; Kanegasaki et al., (2014) Cancer Res 74:5070-5078;Wittrup (2017) Trends Cancer Res 3:615-620.

To evaluate the ability to express collagen-binding chemokines inmammalian cells, three His-tagged collagen-binding cytokines comprisinglumican fused to CCL3, CCL4 or CCL5 were transiently expressed in humanembryonic kidney 293 (HEK293) cells. Briefly, HEK293 cells (at 1 millioncells/mL density) were transfected with sterile-filtered plasmid DNA (1mg per liter cell culture) using polyethylenimine (2 mg per liter cellculture) in OptiPro serum-free media (20 mL per liter cell culture)(Thermo Fisher). TA99 was purified using rProtein A Sepharose Fast Flowresin (GE Healthcare) as previously described (Zhu et al. 2015).His-tagged proteins were isolated from HEK293 supernatant using TALONMetal Affinity Resin (Takara Bio Inc.). Cytokine-fusion proteins werethen further purified by size exclusion chromatrography using a HiLoad16/600 Superdex 200 pg column on an AKTA FPLC system (GE Healthcare)that had been pretreated for 4 hours with 1 M NaOH to remove endotoxinand subsequently equilibrated in sterile PBS (Corning). Followingpurification, all proteins were buffer exchanged into sterile PBS(Corning), 0.2 micron sterile-filtered (Pall Corporation) and confirmedto contain minimal levels of endotoxin (<0.1 EU per injection) using anchromogenic LAL assay (Lonza). To confirm their molecular weights,proteins were run alongside a Novex Prestained Sharp Protein Ladder on a4-12% NuPAGE Bis-Tris protein gel (Life Technologies) with 1% MESrunning buffer. The relative expression levels of the His-taggedcollagen-binding fusion proteins in the resulting eluates was evaluatedby SDS-PAGE (not shown).

Transient expression of Lumican, Lumican D213A (SEQ ID NO: 125),Lumican-Gluc, Lumican-CCL3 (SEQ ID NO: 153), Lumican-CCL4 (SEQ ID NO:156), and Lumican-CCL5 (SEQ ID NO: 160) in HEK293 cells was achieved, asdetermined by SDS-PAGE analysis showing by presence of protein stainingat or near the respective expected molecular weight of each fusionprotein.

These results demonstrate that collagen-binding fusion proteinscomprising chemokines are able to express and be purified from mammaliancells.

To evaluate the ability of the lumican-chemokine fusion proteinsgenerated as described above to induce inflammation (e.g., immune cellmigration), an in vivo inflammatory peritonitis assay was performed aspreviously described (Proudfoot et al., (2003) Proc Natl Acad Sci USA100:1885-1890. Briefly, the peritoneal cavity is lined by a collagen-and vasculature-rich mesothelium. When injected intraperitoneally,matrix-binding constructs (e.g. collagen-binding chemokine fusionproteins) adhere to this lining. In the case of lumican-chemokines,mesothelial localization can create a concentration gradient thatmediates immune cell extravasation out of nearby blood vessels and intothe peritoneal cavity. These infiltrates are retrieved byintraperitoneal lavage for ex vivo immunophenotyping. Accordingly,BALB/c mice were injected intraperitoneally with 1 nmol equivalent in200 uL sterile PBS of either Lumican-Gluc, Lumican-CCL3, Lumican-CCL4,or Lumican-CCL5 and sacrificed 18 hours post-injection. The peritonealcavity was washed three-times by gently massaging 5 mL of ice cold PBSin the cavity and the pooling the lavages. The collected cells wereenumerated using Accuri Flow Cytometer.

Lumican-chemokine fusion proteins comprising CCL3, CCL4, or CCL5 werecapable of mediating overall cell infiltration compared to an injectionof Lumican-GLuc (data not shown). When infiltrates were immunophenotypedby surface marker staining, lavages from mice treated with Lumican-CCL3,Lumican-CCL4 or Lumican-CCL5 contained a greater abundance ofmacrophages, followed by neutrophils, NK cells, DCs, B cells, and Tcells relative to lavages from mice treated with PBS or Lumican-Gluc(data not shown).

These results demonstrate that i.p. administration of collagen-bindingchemokines (e.g., Lumican-CCL3, Lumican-CCL4 or Lumican-CCL5) induceimmune cell migration, including T cells, into the peritoneal cavity ofmice. These results suggest that intratumoral administration oflumican-chemokine fusion proteins will induce inflammation (e.g., immunecell infiltration), thereby mimicking immune-responding tumors.

To assess the therapeutic effect of inducing inflammation usinglumican-chemokines in a tumor setting, Lumican-CCL3 (Lum CCL3) andLumican-CCL5 (Lum CCL5) were tested in both the 4T1 breast tumor modeland B16F10 melanoma model as described herein. Intratumoral treatment oftumor-bearing mice with Lumican-GLuc (20 ug/dose) or a combination ofLumican-CCL3 (5.5 ug/dose), Lumican-CCL4 (5.4 ug/dose) and Lumican-CCL5(5.5 ug/dose) in the presence or absence of IFNα occurred on day 7 andday 13. Intraperitoneal administration of IFNα (50 ug/dose) occurred onday 9 and day 15.

As shown in FIG. 14A, intratumoral injection of a combination ofLumican-CCL3, Lumican-CCL4 and Lumican-CCL5 in the presence of systemicIFNα slightly delayed 4T1 tumor growth. The same treatment administeredin B16F10 tumor-bearing mice conclusively showed that IFNα andlumican-chemokines, administered separately or in combination, delaysgrowth equivalently (FIG. 14B). To determine if lumican-chemokines aredirectly impact the viability of tumor cells, increasing concentrationsof fusion proteins Lumican-GLuc (Lum GLuc), Lumican-CCL3 (Lum CCL3),Lumican-CCL5 (Lum CCL5) were incubated with 4T1 cells or B16F10 in cellculture for 48 hours. After an additional 4 hour incubation with 10 uLof the cell proliferation detection reagent WST-1 (Sigma Aldrich),proliferation was measured by absorbance at 450 nm (reference 700 nm).As shown in FIGS. 14C and 14D, lumican cytokines had no effect onproliferation on 4T1 cells or B16F10 cells indicating any tumor growthdelay observed in vivo arose from an immune-mediated and not from directtumoricidal effect.

Example 18: Synergistic Effect of Collagen-Binding CCL11 ChemokineFusion Protein and Cancer Vaccine in a Mouse Melanoma Tumor Model

Eosinophils are known to secrete T-cell chemoattractants and normalizetumor vasculature thereby easing intratumoral infiltration. Carretero,et al., (2015) Nat Immunol 16:609-617. The chemokine CCL11 (eoxtaxin) isknown to recruit eosinophils (Menzies-Gow et al., (2002) J Immunol169(5):2712-2718). Accordingly, a CCL11-lumican fusion protein (SEQ IDNO: 172), in combination with the cancer vaccine described in Example 14was tested for its ability to recruit eosinophils into a tumor andthereby mediate subsequent tumor control in the presence of systemicTNFα and IFNγ. Briefly, C57BL/6 mice were inoculated with 3×10⁵ B16F10cells into the right flank on day 0. Vaccinations were administeredsubcutaneously (s.c.) at the tail base with a prime on day 5 and boostson day 11 and 17. The vaccination was comprised of 90 ug of TTR-Trp1-EGPand 50 ug of cyclic di-nucleotide to prime a tumor-specific CD8+ T cellresponse. Intratumoral treatments with CCL11-lumican (5 ug/dose/mouse),TNFα (5.8 pmol/dose/mouse) and IFNγ (6.3 pmol/dose/mouse) wereadministered on days 11, 17, 23, and 29. Tumor area (mean+SD) wasmeasured over time every other day

As shown in FIG. 15, treatment of tumor-bearing mice with CCL11-Lumicanin combination with a B16F10-specific cancer vaccine TNFα and IFNγdecreased tumor area to a greater extent than the cancer vaccine aloneor in combination with systemic TNFα and IFNγ.

Example 19: Synergistic Effect of Collagen-Binding CCL11 ChemokineFusion Protein and Cancer Vaccine in a Mouse Melanoma Tumor Model

To further evaluate the ability of a collagen-binding chemokine fusionprotein to synergistically potentiate anti-tumor treatments, theanti-tumor effect(s) of the collagen-binding chemokine fusion proteinCCL11-lumican in combination with MSA-IL2 (30 μg/dose) and atumor-targeting antibody targeting Alpha-V class of integrins (2.5F-Fc;see Kwan et al., (2017) J Exp Med 215(9):10.1084/jem.20160831) wasassessed in an B16-F10 mouse melanoma model. Briefly, 0.5×10⁶ B16-F10cells (ATCC), resuspended in 50 μL of sterile PBS, were inoculatedsubcutaneously into the right flank of 6-8 week old C57BL/6 female miceon day 0. Tumor-targeting antibody 2.5F-Fc and MSA-IL2 were administeredintraperitoneally (i.p.) on days 5, 11, and 17. Intratumoral treatmentswith CCL11-lumican (5 ug/dose/mouse) was given on days 5 and 11. Tumorgrowth was monitored over time every other day and is shown asindividual tumor areas (mm²) in FIG. 16A.

As shown in FIG. 16B, treatment of tumor-bearing mice with CCL11-lumicanin combination with tumor-targeting antibody 2.5F-Fc and MSA-IL-2decreased tumor area to a greater extent than treatment without CCL11(lumican alone).

Collectively, the results shown in Examples 17, 18 and 19 show thatlumican-chemokines induce localized inflammation, recruiting immunecells to the site of local administration and that lumican-chemokinescan be used in combination with other anti-tumor therapeutic modalitiesto potentiate their effects and provide synergistic tumor control.

Example 20: Recombinant Expression of Collagen-Binding Antibody FusionProteins

To evaluate the ability to express collagen-binding antibody fusionproteins in mammalian cells, lumican fused to 5 different antibodieswere generated (4420-Lumican (SEQ ID NOs: 142 and 143;anti-fluorescein), LOB12.3-Lumican (SEQ ID NOs: 146 and 147;anti-4-1BB), 3/23-Lumican (SEQ ID NOs: 184 and 185; anti-CD40),2C11-Lumican (SEQ ID NOs: 150 and 151; anti-CD3), and OX86-Lumican (SEQID NOs: 148 and 149; anti-OX40) and transiently expressed in humanembryonic kidney 293 (HEK293F) cells. All IgG-Lumican fusions wereencoded on a single plasmid (data not shown). All IgG-lumican constructswere expressed as the light chain first (VL), with a murine kappaconstant region (mK), followed by a T2A peptide (SEQ ID NO: 152; T2A)and finally the heavy chain (VH), with a murine IgG2c constant region(mIgG2c), fused to lumican (LUM) with a short linker ((G4S)₃). The T2Apeptide causes the ribosome to skip bond formation between the last tworesidues of the peptide, allowing for expression of two differentproteins within a single open reading frame. A furin cleavage site (F)was included upstream of the T2A peptide, allowing the T2A peptide to beremoved from the end of the light chain. Additionally, a GSG linker(GSG) was included upstream of the T2A peptide, which has been shown toincrease the cleavage efficiency of the T2A peptide (Chng et al., (2015)mAbs 7(2):403-412). Both the light chain and heavy chain comprise aleader sequence to ensure proper trafficking of the protein to thesecretory pathway. All constructs were derived with LALA-PG effectorfunction silencing mutations, which ablate both binding to Fc gammareceptors and binding of C1q (Lo et al., (2017) J Biol Chem292:3900-3908).

Expression and purification of the anti-fluorescein antibody (4420)alone or fused to lumican was achieved as indicated by SDS-PAGE analysisrevealing protein bands located at the predicted molecular weight underboth reducing (R) and non-reducing conditions (NR) (data not shown).Similarly, expression of agonist IgG-lumican fusion proteinsLOB12.3-Lumican (anti-4-1BB), 3/23-Lumican (anti-CD40), 2C11-Lumican(anti-CD3), and OX86-Lumican (anti-OX40) was achieved as indicated bySDS-PAGE analysis revealing protein bands located at the predictedmolecular weight under both reducing (R) and non-reducing conditions(NR) (data not shown). All IgG-lumican fusion proteins were purifiedusing recombinant protein A resin (rProtein A Sepharose, Fast Flow resin(GE Healthcare) according to the manufacturer's recommendations.

These results demonstrate that collagen-binding antibody fusion proteins(e.g., IgG-lumican) express in mammalian cells and that thecollagen-binding polypeptide fusion does not affect purification.

Example 21: Recombinant Collagen-Binding Antibody Fusion Proteins BindCollagen In Vitro and are Retained Intratumorally In Vivo

To evaluate the ability of collagen-binding antibody fusion proteins tobind collagen, the IgG-lumican fusion proteins expressed and purified asdescribed in Example 20 were tested for their ability to bind tocollagen I-coated plates by ELISA. Briefly, collagen I (Gibco) coated 96well-plates were blocked at room temperature for 1 hour with PBS+1%wt/vol bovine serum albumin (BSA)+0.05% wt/vol Tween-20 (PBSTA) and thenincubated with various concentrations of lumican in PBSTA for 2 hours atroom temperature. Wells were washed with PBSTA and then incubated with ahorseradish peroxidase-conjugated goat anti-mouse IgG2c heavy chain(ab98722, Abcam) at a 1:1000 dilution (0.5 μg/ml final concentration) inPBSTA for 1 hour at room temperature. Wells were washed again with PBSTAand then 1-Step Ultra TMB-ELISA Substrate Solution (Thermo FisherScientific) was added for 10 mins followed by 1 M sulfuric acid to stopthe chromogenic reaction. Absorbance at 450 nm (corrected with areference absorbance at 570 nm) measured using an Infinite M1000microplate reader (Tecan). Purified collagen-binding antibody fusionproteins LOB12.3-Lumican (anti-4-1BB), 3/23-Lumican (anti-CD40),2C11-Lumican (anti-CD3), and OX86-Lumican (anti-OX40) were evaluated byELISA on a collagen I-coated plate. As shown in FIG. 17A, allIgG-lumican fusion proteins retain the ability to bind collagen withsimilar measured affinities. These results demonstrate that lumicanfused to the heavy chain of IgGs retains the ability to bind to collagenI.

The ability of the 4420-Lumican fusion protein and the 4420 antibody tobe intratumorally retained in vivo was also evaluated. Both proteinswere purified using size exclusion chromatography then labeled withNHS-AlexaFluor 647 (Thermo Fisher) according to the manufacturer'sinstructions. Six to eight week old female BALB/c mice were injectedwith 1×10⁶ 4T1 mammary carcinoma cells subcutaneously on day 0. On day 7equimolar amounts of fluorescently-labeled 4420 antibody and4420-Lumican were injected intratumorally into three mice each, alongwith three PBS control mice. Retention in the tumor was evaluated viameasuring fluorescence on an IVIS Spectrum instrument (Perkin Elmer) at0, 0.5, 1, 2, 4, 6, 12, 24, 48, 72, 96, 100, 124, and 148 hours (FIG.17B).

As shown in FIG. 17B, the fluorescent signal from mice injected with thefluorescently-labeled 4420 antibody (4420 LALA-PG) decreased faster andto a greater extent than the signal from the 4420-lumican fusion protein(4420-LUM LALA-PG)

These results demonstrate that collagen-binding antibody fusion proteins(e.g., 4420-lumican fusion protein) are physically retained at the siteof intratumoral injection over time. These results suggest thatcollagen-binding immunomodulatory molecules comprising a therapeuticantibody or antigen-binding fragment will exhibit intratumoral retentionand limited systemic dissemination.

Example 22: Recombinant Expression of Collagen-Binding IgG-BindingFusion Proteins

As a strategy to localize virtually any IgG intraumorally withoutrequiring regenerating the antibody as a direct lumican fusion, severaldifferent IgG binding proteins were fused to lumican. As with otherlumican fusion proteins described herein, a mouse serum albumin (MSA)spacer was used to ensure that lumican-collagen binding did notinterfere with the functionality of the IgG binding domain. Severaldifferent IgG binders were selected for screening, including a dimerizedZ domain (one of the five IgG binding domains of protein A, hereinreferred to as “ZZ”) (Jendeberg et al., (1995) J Mol Recognit8:270-278), a dimerized IgG binding domain of protein G (herein referredto as “SpG2”) (Jung et al., (2009) Anal Chem 81:936-942), an IgG binderisolated from a Sso7d yeast display library (Gera et al., (2011) J MolBiol 409” 601-616), an IgG binder isolated from a Fibronectin type IIIdomain (Fn3) yeast display library (Hackel et al., (2010) J Mol Biol401:84-96), and two small peptides designed to bind IgG Fc regions(herein referred to as “Fc-III-4C” and “RRGW) (Gong et al., (2015)Bioconjug Chem 27:1569-1573; Tsai et al., (2014) Anal Chem86:2931-2938). In addition, a lumican-MSA fusion to 4m5.3 was alsocloned and expressed. 4m5.3 is an scFv with femtomolar binding affinityto fluorescein (Midelfort et al., (2004) J Mol Biol 343:685-701).Fluorescein can be conjugated to antibodies with a wide range ofcoupling strategies borrowed from the field of antibody drug conjugates(ADCs) (Carter & Lazar (2017) Nat Rev Drug Discov 17:197-223. Using4m5.3-MSA-Lumican with fluorescently labeled antibodies serves as analternative strategy for localizing IgGs to the tumor. This constructalso serves as a generalized platform ably to tightly bind to andlocalize any fluorescein (or FITC) labeled protein or small molecule.

To evaluate the ability to express collagen-binding IgG-binding fusionproteins in mammalian cells, lumican fused to 8 different IgG-bindingpolypeptides were generated (Lumican-MSA-Fc-III-4C (SEQ ID NO: 136;105.7 kDa), Lumican-MSA-Fn3 (SEQ ID NO: 137; 113.7 kDa),Lumican-MSA-SpG2 (SEQ ID NO: 138; 117.7 kDa), ZZ-MSA-Lumican (SEQ ID NO:135; 117.5 kDa), WGRR-MSA-Lumican (SEQ ID NO: 140; 104.6 kDa),RRGW-MSA-Lumican (SEQ ID NO: 139; 104.6 kDa), Sso7d-MSA-Lumican (SEQ IDNO: 134; 111.5 kDa), and 4m5.3-MSA-Lumican (SEQ ID NO: 133; 132.2 kDa))and transiently expressed in human embryonic kidney 293 (HEK293) cells.All lumican-IgG binding fusion proteins were His-tagged to facilitatepurification from HEK293 lysates using TALON Metal Affinity Resin(Takara Bio Inc.) according to the manufacturer's instructions.

Expression and purification of all 8 lumican-IgG-binding fusion proteinswas achieved as indicated by SDS-PAGE analysis revealing protein bandslocated at the predicted molecular weights under both reducing andnon-reducing conditions (data not shown).

These results demonstrate that His-tagged collagen-binding IgG-bindingfusion proteins (e.g., IgG-lumican) express in mammalian cells and areable to be purified.

Example 23: Recombinant Collagen-Binding IgG-Binding Fusion ProteinsBind Collagen and IgG In Vitro

To evaluate the ability of collagen-binding IgG-binding fusion proteinsto bind collagen, the lumican-IgG binding fusion proteins expressed andpurified as described in Example 22 were tested for their ability tobind to collagen I- and collagen IV-coated plates by ELISA. Briefly,Nunc MaxiSorp flat bottom 96 well-plates (ThermoFisher) were coatedovernight with mouse IgG2a isotype control antibodies (100 μL, 2.5μg/mL, BioXCell C1.18.4) overnight at 4 C. Plates were then blocked atroom temperature for 1 hour with PBS+1% wt/vol bovine serum albumin(BSA)+0.05% wt/vol Tween-20 (PBSTA) and then incubated with variousconcentrations of lumican in PBSTA for 2 hours at room temperature.Wells were washed with PBSTA and then incubated with a horseradishperoxidase-conjugated polyclonal anti-6×His (ab1187, Abcam) at a 1:2000dilution (0.5 μg/ml final concentration) in PBSTA for 1 hour at roomtemperature. Wells were washed again with PBSTA and then 1-Step UltraTMB-ELISA Substrate Solution (Thermo Fisher Scientific) was added for 10mins followed by 1 M sulfuric acid to stop the chromogenic reaction.Absorbance at 450 nm (corrected with a reference absorbance at 570 nm)measured using an Infinite M1000 microplate reader (Tecan).

The purified lumican-IgG-binding fusion proteins Lumican-MSA-Fn3,Lumican-MSA-SpG2, ZZ-MSA-Lumican, and 4m5.3-MSA-Lumican were evaluatedby ELISA on a collagen I-coated plate and a collagen IC-coated plate.Lumican was used as a comparator. As shown in FIG. 18A, alllumican-IgG-binding fusion proteins retain the ability to bind collagenwith similar measured affinities. These results demonstrate that lumicanfused to various IgG-binding polypeptides retains the ability to bind tocollagen I and collagen IV.

The purified lumican-IgG-binding fusion proteins from Example 22 weretested for their ability to bind mouse IgG2a isotype control (CloneC1.18.4) as measured via ELISA. Briefly, an anti-His secondary antibody(Abcam, ab1187) conjugated to HRP was used to detect the IgGbinder-lumican fusions, along with 1-step Ultra TMB-ELISA Substrate(Thermo Fischer).

As shown in FIG. 18B, all lumican-IgG-binding fusion proteins testedbind to mouse IgG2a, with a range of affinities (K_(D)).

Collectively, these results demonstrate that lumican-IgG-binding fusionprotein bind to both collagen I and collagen IV and bind to IgGs. Theseresults suggest that lumican-IgG-binding fusion proteins used incombination with IgG (e.g., therapeutic antibodies) would bind to bothcollagen and IgG and retain the IgG at the site of local administration.

Example 24: Collagen-Binding Lumican is Retained in the PeritonealCavity Following Intraperitoneal Injection

The aforementioned Examples have demonstrated the utility of lumican tobe retained intratumorally upon intratumoral (i.tu.) administration. Theperitoneal cavity is also lined by a collagen-rich mesothelium. Toevaluate the ability of lumican to be retained in the peritoneal cavityupon intraperitoneal (i.p) injection, BALB/c mice were injectedintraperitoneally with either Gaussia Luciferase (GLuc; 20 μg/dose)alone or fused to Lumican (Lumican-Gluc; 40 μg/dose). Immediately afterthe injection, mice were imaged by in vivo fluorescence(epi-illumination, auto-exposure). 24 hours after the injection, micewere imaged again.

Lumican-GLuc is retained in the cavity however the GLuc alone rapidlydiffuses in the peritoneum immediately following injection, leading tolow initial signal (data not shown). Retention of Lumican was observedin the cavity 24 hours post-injection.

Tumors embedded on this lining or the omentum of the peritoneal cavityare also collagen-rich as well. To determine if i.p. administration oflumican will result in the accumulation on tumors liking the omentum,lumican fluorescently labeled with Alexa Fluor 647 was administered tomice having ovarian tumor microcolonies in the mouse omental tissue.Briefly, mice were injected with OVCA433 cells a human ovarian tumorcell line intraperitoneally and allowed to form microcolonies on theomentum for three weeks. 20 ug of labeled lumican was injectedintraperitoneally. Excised omentum tissue was imaged by fluorescencemicroscopy. Labeled lumican was injected intraperitoneally intumor-bearing mice. At 1 hour, 6 hours and 24 hours post-injection,omentums of mice were excised for imaging.

As shown in FIG. 19, when Lumican is intraperitoneally injected intomice bearing ovarian tumors lining the omentum, lumican preferentiallyaccumulates at these tumor microcolonies. One hour post injection (leftpanel), the fluorescent signal from lumican (shown in yellow) wasdistributed uniformly. After 6 hours post-injection (middle) it waslargely retained only around tumor microcolonies (shown in red) in theomentum (middle panel). Retention of the lumican was observed at 24hours (right panel) around large tumors. These results demonstrate thati.p. administration of lumican can accumulate on collagen-rich locationsand that lumican is amenable to several modes of administrationincluding intraperitoneal injection.

Example 25: Expression of Collagen-Binding IL-12 Fusion Proteins fromSelf-Replicating RNA in Mammalian Cells

To evaluate the expression of collagen-binding immunomodulatorymolecules using RNA, self-replicating RNA molecules (replicons) encodingIL12-MSA and IL12-MSA-Lumican alone or fused to a fluorescent protein(mCherry) were tested for their ability to express in B16F10 mousemelanoma cells. The replicon used in this Example was derived from analphavirus. The replicon was deleted of capsid structural proteins, butretains the non-structural proteins. The non-structural proteinscorrespond to the RNA-based RNA replication and RNA transcription. TheAb1c1 is a mutant replicon that exhibits more robust expression in vitroand in vivo compared to the wild-type replicon. The Ab1c1 repliconcontains four mutations in the nsP2 and nsP3 genes that prolong repliconexistence in cells and subgenomic transcription.

Briefly, 0.5×10⁶ B16F10 cells cultured in DMEM+10% FBS were transfectedusing NEOnN transfection reagents (electroporation) and the Ab1c1replicons. The transfection efficiency was detected by FACS analyzer 24hours later using optical settings to detect mCherry. Expression ofIL-12 was quantified using cell supernatant 24 hours after thetransfection and a commercial IL-12 ELISA (followed according tomanufacturer's instructions). Expression of replicons was evaluated inB16F10 cells by determination of mCherry by flow cytometry (FIG. 20A). Acommercial IL-12 ELISA was used to quantify IL-12 in B16F10 cell (FIG.20B).

As shown in FIG. 20A, mCherry+B16F10 cells transfected withIL12-MSA-mCherry or IL12-MSA-Lumican-mCherry were observed by flowcytometry. The mCherry fluorescent protein is C-terminally encoded inthese replications, therefore, detection of mCherry expression isindication of full-length expression of the encoded protein.

As shown in FIG. 20B, cell supernatants shows expression of theseconstructs from B16F10 cells transfected with IL-12-encodingreplications, but not the Ab1c1-GIM control

These results demonstrate that delivery of Lumican-fusion proteins isachievable using modes of administration beyond injection, includingreplicon as shown.

Example 26: Synergistic Effect of Collagen-Binding Cytokine FusionProteins, anti-PD-1 Antibody and TA99 in the Braf^(v600e)/Pten^(fl/fl)Mouse Model

To evaluate the effectiveness of lumican-cytokines in combination withanti-PD-1 blockade in a difficult-to-treat mouse melanoma model, theBraf^(V600E)/Pten^(fl/fl) genetically modified mouse model (GEMM)(Spranger et al., (2015) Nature 523:231-235; Momin et al., (2019) Sci.Transl. Med. 11, eeaw2614). Melanomas in this GEMM are induced bytamoxifen-regulated Cre expression in melanocytes, which drives theactivation of oncogenic Braf^(V600E) and biallelic deletion of tumorsuppressor Pten. Braf^(V600E)/Pten^(fl/fl) melanomas have fewerneoantigens and more heterogeneity than B16F10 tumors. This model hasmodest T cell infiltration, but tumor growth is only slightly slowed bydual checkpoint blockade of PD-1 and cytotoxic T-lymphocyte-associatedprotein 4 (CTLA-4). Id.

To determine if treatment with collagen-binding IL-2 and IL-12 alongsideanti-PD-1 checkpoint blockade could reinvigorate the ongoing responseand prime new T cell responses, as was observed in B16F10 tumors (FIGS.9, 10, and 13), and if de novo T cell priming and any abscopal effectscould be augmented by the inclusion of an immunogenic celldeath-directing agent like the tumor-targeting antibody TA99, melanomawas induced by application of 4-hydroxytamoxifen on the right flank ofBraf^(V600E)/Pten^(fl/fl) mice on day 0. After induction, flatblack-pigment lesions are formed. The lesions will progress to largemasses without therapy (data not shown). On day 25, the tumor-bearingmice are treated with PBS control (i.tu), with Lumican-MSA-IL2(i.tu)+IL12-MSA-Lumican (i.tu)+TA99 (i.p.)+anti-PD-1 (i.p.), withLumican-MSA-IL2 (i.tu)+IL12-MSA-Lumican (i.tu)+anti-PD-1 (i.p.), withMSA-IL2 (i.tu)+IL12-MSA (i.tu)+TA99 (i.p.)+anti-PD-1 (i.p.), or withMSA-IL2 (i.tu)+IL12-MSA(i.tu)+anti-PD-1 (i.p.) on days 25, 31, 37, 43,49, 55, and 61.

As shown in FIG. 21A, Braf^(V600E)/Pten^(fl/fl) tumor-bearing micetreated with MSA-IL2 (i.tu)+IL12-MSA (i.tu)+TA99 (i.p.)+anti-PD-1 (i.p.)display inhibited lesion development.

As shown in FIG. 21B, the overall survival of Braf^(V600E)/Pten^(fl/fl)tumor-bearing mice treated with anti-PD-1 and collagen-binding IL-2 andIL-12 with or without TA99 is comparable. Thus, TA99 was not a necessarycomponent for efficacy. These results affirm that IL-2, IL-12, andcheckpoint blockade can be an effective tumor-agnostic combinationtreatment (FIGS. 9, 10 and 13).

Tumor control in this model could also be achieved using unanchoredcytokines in lieu of collagen-anchoring cytokines, but at the cost ofmajor and potentially lethal toxicity. One-third of the mice treatedwith IL12-MSA and MSA-IL2 were euthanized because of >20% loss of bodyweight, whereas no mice treated with IL12-MSA-Lumican andLumican-MSA-IL2 succumbed to treatment-related toxicity (FIG. 21B).These results demonstrate that collagen binding fusion proteins cansafely improve overall survival in this potent tumor-agnosticcombination treatment (FIG. 21B).

Example 27: LAIR Capacity for Binding to In Vivo Tumors

In this example, the capacity of LAIR to bind excised B16F10 tumors ismeasured. B16F10 tumors possess little detectable collagen and thereforeserves as a lower estimate for LAIR's binding-capacity to a tumor.Briefly, C57/mice were inoculated with 1×10⁶ B16F10 cells injectedsubcutaneously into the left flank. Seven days later, tumors werecarefully excised and detached from all remaining skin and subcutaneousfat. The excised tumors were then incubated in a gentle detergent(PBS+0.1% v/v Tween20) for 2 hours at 37° C. and thendisaggregated/pushed through a 70 micron filter (FIG. 22A). As shown inFIG. 22B, the matrix comprises a third of the tumor's mass.

The filtered-fraction was devoid of extracellular matrix (i.e. cellfraction) while the fraction that did not pass the filter (i.e. matrixfraction) was matrix-rich, as confirmed by a hydroxyproline assay(MAK008-1KT, Millipore Sigma) performed according to the manufacturer'sinstructions (FIG. 22C). Hydroxyproline (4-hydroxyproline) is anon-proteinogenic amino acid formed by the post-translationalhydroxylation of proline. Hydroxyproline is a major component ofcollagen, where it serves to stabilize the helical structure. Becausehydroxyproline is largely restricted to collagen, the measurement ofhydroxylproline levels is used as an indicator of collagen content. Inthis assay, hydroxyproline concentration is detected by the reaction ofoxidized hydroxyproline with 4-(dimethylamino)benzaldehyde (DMAB), whichresults in development of a colorimetric (560 nm) product, proportionalto the hydroxyproline present.

Each tumor's matrix fraction was then incubated in either anantigen-excess concentration (10 μM) or antigen-depleting concentration(10 μM) of AF647-labeled LAIR to quantitate LAIR binding sites in thematrix fraction. The fluorescence of the solution was monitored overtime until steady-state is achieved. As shown in FIG. 22D, a decrease influorescence is observed with the 1 μM labeled LAIR, which correspondsto LAIR depletion from the bath upon binding to the collagen within thematrix fraction. A 20% decrease in solution concentration indicates 0.2nmol uptake into the tumor matrix. Thus, a day 7 B16F10 tumor from a1×10⁶ cell inoculum possesses 0.2 nmol of LAIR binding sites (FIG. 22D).This number correlates to the tumor's collagen content, as measured byhydroxyproline content (FIG. 22E). This experiment shows that LAIR iscapable of binding B1610, as the B1610 matrix is collagen rich.

Example 28: Collagen-Binding Fusion Proteins Using LAIR Imparts SimilarBenefit Compared to Using Lumican

Like Lumican, LAIR can bind to collagen type I. A LAIR cytokine fusionprotein was used to determine whether the collagen-binding fusionprotein could potentiate the efficacy of a fused cytokine. ALAIR-cytokine fusion protein, LAIR-MSA-IL2 (SEQ ID NO: 186), wasexpressed and purified as described for Lumican-MSA-IL2 (Example 1). Inthe B16F10 melanoma mouse model conducted as described in Example 6,LAIR-MSA-IL2 is at least as efficacious as Lumican-MSA-IL2 in reducingtumor size (compare FIG. 3B to FIG. 23A). LAIR-MSA IL-2 also increasedmice survival to levels comparable to survival in mice treated with acombination of Lumican-MSA-IL2 with intraperitoneal TA99 (compare FIGS.3B and 3C to FIG. 23B). These results demonstrate that thecollagen-binding strategy can be utilized using Lumican, LAIR or othercollagen-binding proteins in their class.

Example 29: LAIR-Engineering Yields Both Higher and Lower AffinityCollagen Binders

In this example, a yeast display platform is utilized to engineer higherand lower affinity variants of LAIR. Briefly, the mouse LAIR gene wasamplified using error prone PCR to produce a library of LAIR mutants.RJY200 yeast (in-house modified version of EBY100, ATCC) are transformedwith linearized pCTCON2 vector (41843, Addgene) and the error prone PCRproduct is collected, and subjected to an in vivo homologousrecombination event to produce the final display plasmid. The pCTCON2plasmid is formatted such that the LAIR mutants are fused to the Aga2protein, which is bound to the yeast surface via a disulfide linkage tothe membrane bound Aga1 protein. The LAIR gene is also followed by ac-myc tag, allowing one to probe for full expression of the mutant LAIRprotein. Once expression is induced on the surface, yeast can be stainedwith labeled antigen and an antibody against the c-myc tag (ACMYC,Exalpha). LAIR expressing clones, as determined by c-myc stainingintensity, can then be sorted for lower or higher affinity using FACS.(Chao et al., (2006) Nat. Protoc. 1(2):755-768).

A soluble collagen peptide mimic (CRP, collagen related peptide) wasused as the antigen for the FACS assay as the LAIR natural ligand,collagen I, is not soluble. The protein sequence for this mimic isGCO-(GPO)₁₀-GCOG-NH2 (SEQ ID NO: 212) where O represents hydroxyprolineamino acids. Similar to collagen I, these peptides spontaneously formhelical structures in solution. A crosslinking reagent (cat #, company)was used to lock these helical structures in place. Followingpurification, the crosslinked peptide was used as our antigen (CRP-XL).Note that this peptide was used in both a biotinylated (CRP-XL-Biotin)and non-biotinylated (CRP-XL) format. (A detailed description on thesepeptides and their functionalization into a triple helix format can beobtained from CambCollab Inc.)

Two different strategies were used to isolate high and low affinitycollagen-binding mutants. To select for low affinity mutants,equilibrium sorting was employed. In this strategy, LAIR expression wasinduced on the surface of the yeast library. The library wassequentially incubated with CPP-XL-biotin and chicken anti-c-myc (ACMYC,Exalpha), followed by secondary antibodies (streptavidin-AF647 (S21374,Thermo Fisher) and goat anti-chicken AF488 (A-1139, Invitrogen) untilequilibrium was reached. Yeast that displayed weak or no AF647 signalbut were positive for AF488, indicating that they were expressing LAIRbut not binding to the collagen mimic, were sorted on a BD FACS Ariamachine. After several rounds of sorting, the yeast were miniprepped toisolate the display plasmids, transformed into bacteria colonies, andsubmitted for sequencing. Prevalent clones (clones that appeared insequencing at higher frequency than others (at least twice)) and/orclones that contained mutations in the collagen binding pocket wereselected for downstream analysis. (Brondijk et al., (2010) Blood115:1364-1373). These mutants were cloned into a mammalian expressionvector, solubly expressed as fusions to mouse serum albumin (MSA), andtested for their ability to bind collagen I in an ELISA assays. As shownin FIGS. 24A-E and 25A-C, weakly binding clones (FIGS. 24F, 24D) containmutations in the LAIR binding pocket. SEQ ID NOS: 187-192.

To isolate higher affinity mutants, a kinetic sort strategy wasemployed. After inducing expression, the yeast library was labeled withCRP-XL-biotin as described above. After equilibrium was reached, theyeast clones were washed and then incubated in 300:1 excess CRP-XL(non-biotinylated) for 3-5 days. Unlabeled CRP-XL will displacedissociated CRP-XL-biotin for LAIR binding (FIGS. 26C-D). Only theclones with the slowest off-rate, i.e., the highest affinity clones,will remain labeled. The yeast clones with highest AF647 signal werethen sorted using FACS. After several rounds of sorting, high affinityclones were selected for downstream analysis in a competition assay. Theisolated yeast clones were labeled with CRP-XL-biotin and then incubatedwith unlabeled CRP-XL. Samples were taken over time and analyzed forAF647 signal (FIGS. 26E-F). As shown in FIG. 26G, a high affinity themutant LAIR, LAIR30.2.K1.B, has a slower off rate than WT LAIR. Themutations seen in this clone is located outside of the LAIR bindingpocket (FIG. 26B, SEQ ID NO: 193). These results show that mutant LAIRswith a range of binding affinities to collagen could be isolated. TheseLAIR1 variants provide an opportunity to engineer immunomodulatoryfusion proteins comprising therapeutic agents that are have differentbinding affinities to collagen-rich tumors.

Summary Sequence Table SEQ ID NO Description Sequence 1 IL-2APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT 2 Wild TypeIWELKKDVYVVELDWYPDAPGEMVVLTCDTPEEDGITWTLDQSSEVLGSGKTLTIQ IL12B withoutVKEFGDAGQYTCHKGGEVLSHSLLLLHKKEDGIWSTDILKDQKEPKNKTFLRCEAK signal (IL12B)NYSGRFTCWWLTT1STDLTFSVKSSRGSSDPQGVTCGAATLSAERVRGDNKEYEYS Amino AcidsVECQEDSACPAAEESLPIEVMVDAVHKLKYENYTSSFFIRDIIKPDPPKNLQLKPLKNSRQVEVSWEYPDTWSTPHSYFSLTFCVQVQGKSKREKKDRVFTDKTSATVICRKNASISVRAQDRYYSSSWSEWASVPCS 3 Wild TypeRNLPVATPDPGMFPCLHHSQNLLRAVSNMLQKARQTLEFYPCTSEEIDHEDITKDK IL12A withoutTSTVEACLPLELTKNESCLNSRETSFITNGSCLASRKTSFMMALCLSSIYEDLKMY signal peptideQVEFKTMNAKLLMDPKRQIFLDQNMLAVIDELMQALNFNSETVPQKSSLEEPDFYK Amino acidsTKIKLCILLHAFRIRAVTIDRVMSYLNAS 4 IL-15RaITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPPSTVTTAGVTPQPESLSPSGKEPAASSPSSNNTAATTAAIVPGSQLMPSKSPSTGTTEISSHESSHGTPSQTTAKNWELTASASHQPPGVYP QGHSDTT 5 IL-15NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFIN TS 6 TNF-alphaVRSSSRTPSDKPVAHVVANPQAEGQLQWLNRRANALLANGVELRDNQLVVPSEGLYLIYSQVLFKGQGCPSTHVLLTHTISRIAVSYQTKVNLLSAIKSPCQRETPEGAEAKPWYEPIYLCGVFQLEKGDRLSAEINRPDYLDFAESCQVYFCIIAL 7 IFN-gammaQDPYVKEAENLKKYFNAGHSDVADNGTLFLGILKNWKEESDRKIMQSQIVSFYFKLFKNFKDDQSIQKSVETIKEDMNVKFFNSNKKKRDDFEKLTNYSVFDLNVQRKAIHELIQVMAELSPAAKTGKRKRSQMLFRG 8 IFN-alphaCDLPQTHSLGSRRTLMLLAQMRKISLFSCLKDRHDFGFPQEEFGNQFQKAETIPVLHEMIQQIFNLFSTKDSSAAWDETLLDKFYTELYQQLNDLEACVIQGVGVTETPLMKEDSILAVRKYFQRITLYLKEKKYSPCAWEVVRAEIMRSFSLSTNLQESLRSKE 9 IL-21QGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAQLKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPKEFLERFKSLLQKMIHQHLSSRTHGSEDS 10 IL-6LAPRRCPAQEVARGVLTSLPGDSVTLTCPGVEPEDNATVHWVLRKPAAGSHPSRWAGMGRRLLLRSVQLHDSGNYSCYRAGRPAGTVHLLVDVPPEEPQLSCFRKSPLSNVVCEWGPRSTPSLTTKAVLLVRKFQNSPAEDFQEPCQYSQESQKFSCQLAVPEGDSSFYIVSMCVASSVGSKFSKTQTFQGCGILQPDPPANITVTAVARNPRWLSVTWQDPHSWNSSFYRLRFELRYRAERSKTFTTWMVKDLQHHCVIHDAWSGLRHVVQLRAQEEFGQGEWSEWSPEAMGTPWTESRSPPAENEVSTPMQALTTNKDDDNILFRDSANATSLPVQDSSSVPLPTFLVAGGSLAFGTLLCIAIVLRFKKTWKLRALKEGKTSMHPPYSLGQLVPERPRPTPVLVPLISPPVSPSSLGSDNTSSHNRPDARDPRSPYDISNTDYFFP R 11 IL-5DLLPDEKISLLPPVNFTIKVTGLAQVLLQWKPNPDQEQRNVNLEYQVKINAPKEDDYETRITESKCVTILHKGFSASVRTILQNDHSLLASSWASAELHAPPGSPGISIVNLTCTTNTTEDNYSRLRSYQVSLHCTWLVGTDAPEDTQYFLYYRYGSWTEECQEYSKDTLGRNIACWFPRTFILSKGRDWLAVLVNGSSKHSAIRPFDQLFALHAIDQINPPLNVTAEIEGTRLSIQWEKPVSAFPIHCFDYEVKIHNTRNGYLQIEKLMTNAFISIIDDLSKYDVQVRAAVSSMCREAGLWSEWSQPIYVGNDEHKPLREWFVIVIMATICFILLILSLICKICHLWIKLFPPIPAPKSNIKDLFVGGNYEKAGSSETEIEVICYIEKPGV ETLEDSVF 12IL-8 AVLPRSAKELRCQCIKTYSKPFHPKFIKELRVIESGPHCANTEIIVKLSDGRELCLDPKENWVQRWEKFLKRAENS 13 IL-7DCDIEGKDGKQYESVLMVSIDQLLDSMKEIGSNCLNNEFNFFKRHICDANKEGMFLFRAARKLRQFLKMNSTGDFDLHLLKVSEGTTILLNCTGQVKGRKPAALGEAQPTKSLEENKSLKEQKKLNDLCFLKRLLQEIKTCWNKILMGTKEH 14 IL-17AGITIPRNPGCPNSEDKNFPRTVMVNLNIHNRNTNTNPKRSSDYYNRSTSPWNLHRNEDPERYPSVIWEAKCRHLGCINADGNVDYHMNSVPIQQEILVLRREPPHCPNSFRLEKILVSVGCTCVTPIVHHVA 15 IL-13alphaRAVPGGSSPAWTQCQQLSQKLCTLAWSAHPLVGHMDLREEGDEETTNDVPHIQCGDGCDPQGLRDNSQFCLQRIHQGLIFYEKLLGSDIFTGEPSLLPDSPVGQLHASLLGLSQLLQPEGHHWETQQIPSLSPSQPWQRLLLRFKILRSLQAFVAVAARVFAHGAATL SP 16 IL-18YFGKLESKLSVIRNLNDQVLFIDQGNRPLFEDMTDSDCRDNAPRTIFIISMYKDSQPRGMAVTISVKCEKISTLSCENKIISFKEMNPPDNIKDTKSDIIFFQRSVPGHDNKMQFESSSYEGYFLACEKERDLFKLILKKEDELGDRSIMFTVQNED 17 IL-1alphaSAPFSFLSNVKYNFMRIIKYEFILNDALNQSIIRANDQYLTAAALHNLDEAVKFDMGAYKSSKDDAKITVILRISKTQLYVTAQDEDQPVLLKEMPEIPKTITGSETNLLFFWETHGTKNYFTSVAHPNLFIATKQDYWVCLAGGPPSITDFQILENQA 18 IL-1betaAPVRSLNCTLRDSQQKSLVMSGPYELKALHLQGQDMEQQVVFSMSFVQGEESNDKIPVALGLKEKNLYLSCVLKDDKPTLQLESVDPKNYPKKKMEKRFVFNKIEINNKLEFESAQFPNWYISTSQAENMPVFLGGTKGGQDITDFTMQFVSS 19 IL-4HKCDITLQEIIKTLNSLTEQKTLCTELTVTDIFAASKNTTEKETFCRAATVLRQFYSHHEKDTRCLGATAQQFHRHKQLIRFLKRLDRNLWGLAGLNSCPVKEANQSTLENFLERLKTIMREKYSKCSS 20 IL-3APMTQTTPLKTSWVNCSNMIDEIITHLKQPPLPLLDFNNLNGEDQDILMENNLRRPNLEAFNRAVKSLQNASAIESILKNLLPCLPLATAAPTRHPIHIKDGDWNEFRRKLTFYLKTLENAQAQQTTLSLAIF 21 IL-10SPGQGTQSENSCTHFPGNLPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLRLRLRRCHRFLPCENKSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRN 22 IL-13PVPPSTALRELIEELVNITQNQKAPLCNGSMVWSINLTAGMYCAALESLINVSGCSAIEKTQRMLSGFCPHKVSAGQFSSLHVRDTKIEVAQFVKDLLLHLKKLFREGRFN 23 IL-17aGITIPRNPGCPNSEDKNFPRTVMVNLNIHNRNTNTNPKRSSDYYNRSTSPWNLHRNEDPERYPSVIWEAKCRHLGCINADGNVDYHMNSVPIQQEILVLRREPPHCPNSFRLEKILVSVGCTCVTPIVHHVA 24 IL-9QGCPTLAGILDINFLINKMQEDPASKCHCSANVTSCLCLGIPSDNCTRPCFSERLSQMTNTTMQTRYPLIFSRVKKSVEVLKNNKCPYFSCEQPCNQTTAGNALTFLKSLLE IFQKEKMRGMRGKI25 IFN-gamma QDPYVKEAENLKKYFNAGHSDVADNGTLFLGILKNWKEESDRKIMQSQIVSFYFKLFKNFKDDQSIQKSVETIKEDMNVKFFNSNKKKRDDFEKLTNYSVTDLNVQRKAIHELIQVMAELSPAAKTGKRKRSQMLFRG 26 IFN-alphaCDLPQTHSLGSRRTLMLLAQMRKISLFSCLKDRHDFGFPQEEFGNQFQKAETIPVLHEMIQQIFNLFSTKDSSAAWDETLLDKFYTELYQQLNDLEACVIQGVGVTETPLMKEDSILAVRKYFQRITLYLKEKKYSPCAWEVVRAEIMRSFSLSTNLQESLRSKE 27 GM-CSFAPARSPSPSTQPWEHVNAIQEARRLLNLSRDTAAEMNETVEVISEMFDLQEPTCLQTRLELYKQGLRGSLTKLKGPLTMMASHYKQHCPPTPETSCATQIITFESFKENLKD FLLVIPFDCWEPVQE28 FLT3L TQDCSFQHSPISSDFAVKIRELSDYLLQDYPVTVASNLQDEELCGGLWRLVLAQRWMERLKTVAGSKMQGLLERVNTEIHFVTKCAFQPPPSCLRFVQTNISRLLQETSEQLVALKPWITRQNFSRCLELQCQPDSSTLPPPWSPRPLEATAPTAPQPPLLLLLLLPVGLLLLAAAWCLHWQRTRRRTPRPGEQVPPVPSPQDLLLVEH 29 G-CSFATPLGPASSLPQSFLLKCLEQVRKIQGDGAALQEKLVSECATYKLCHPEELVLLGHSLGIPWAPLSSCPSQALQLAGCLSQLHSGLFLYQGLLQALEGISPELGPTLDTLQLDVADFATTIWQQMEELGMAPALQPTQGAMPAFASAFQRRAGGVLVASHLQSFLEVS YRVLRHLAQP 30LIF SPLPITPVNATCAIRHPCHNNLMNQIRSQLAQLNGSANALFILYYTAQGEPFPNNLDKLCGPNVTDFPPFHANGTEKAKLVELYRIWYLGTSLGNITRDQKILNPSALSLHSKLNATADILRGLLSNVLCRLCSKYHVGHVDVTYGPDTSGKDVFQKKKLGCQLLGK YKQIIAVLAQAF 31M-CSF EEVSEYCSHMIGSGHLQSLQRLIDSQMETSCQITFEFVDQEQLKDPVCYLKKAFLLVQDIMEDTMRFRDNTPNAIAIVQLQELSLRLKSCFTKDYEEHDKACVRTFYETPLQLLEKVKNVFNETKNLLDKDWNIFSKNCNNSFAECSSQDWTKPDCNCLYPKAIPSSDPASVSPHQPLAPSMAPVAGLTWEDSEGTEGSSLLPGEQPLHTVDPGSAKQRPPRSTCQSFEPPETPWKDSTIGGSPQPRPSVGAFNPGMEDILDSAMGTNWVPEEASGEASEIPVPQGTELSPSRPGGGSMQTEPARPSNFLSASSPLPASAKGQQPADVTGTALPRVGPVRPTGQDWNHTPQKTDHPSALLRDPPEPGSPRISSLRPQGLSNPSTLSAQPQLSRSHSSGSVLPLGELEGRRSTRDRRSPAEPEGGPASEGAARPLPRFNSVPLTDTGHERQSEGSFSPQLQESVFHLLVPSVILVLLAVGGLLFYRWRRRSHQEPQRADSPLEQPEGSPLTQDDRQVELPV 32 MIP-2APLATELRCQCLQTLQGIHLKNIQSVKVKSPGPHCAQTEVIATLKNGQKACLNPASPMVKKIIEKMLKNGKSN 33 MIP-1betaAPMGSDPPTACCFSYTARKLPRNFWDYYETSSLCSQPAWFQTKRSKQVCADPSE SWVQEYVYDLELN 34KP (aka CXCL1) ASVATELRCQCLQTLQGIHPKNIQSVNVKSPGPHCAQTEVIATLKNGRKACLNPASPIVKKIIEKMLNSDKSN 35 MIG (aka CXCL9)TPVVRKGRCSCISTNQGTIHLQSLKDLKQFAPSPSCEKIEIIATLKNGVQTCLNPDSADVKELIKKWEKQVSQKKKQKNGKKHQKKKVLKVRKSQRSRQKKTT 36 IP-10 (CXCL10)VPLSRTVRCTCISISNQPVNPRSLEKLEIIPASQFCPRVEIIATMKKKGEKRCLNPESKAIKNLLKAVSKERSKRSP 37 MCP-1QPDAINAPVTCCYNFTNRKISVQRLASYRRITSSKCPKEAVIFKTIVAKEICADPKQKWVQDSMDHLDKQTQTPKT 38 EotaxinGPASVPTTCCFNLANRKIPLQRLESYRRITSGKCPQKAVIFKTKLAKDICADPKKKWVQDSMKYLDQKSPTPKP 39 RANTESSPYSSDTTPCCFAYIARPLPRAHIKEYFYTSGKCSNPAWFVTRKNRQVCANPEKK WVREYINSLEMS 40LIX AGPAAAVLRELRCVCLQTTQGVHPKMISNLQVFAIGPQCSKVEWASLKNGKEICLDPEAPFLKKVIQKILDGGNKEN 41 MIP-1alphaSLAADTPTACCFSYTSRQIPQNFIADYFETSSQCSKPGVIFLTKRSRQVCADPSEE WVQKYVSDLELSA42 Human serum  MDMRVPAQLLGLLLLWLPGARCADAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQalbumin CPFEDHVKLVNEVGEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADamino acid  CCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRsequence) HPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDEGYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGLGGGSAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLGGGGS 43 Mature HSADAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVAD (amino acid ESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNL sequence)PRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGLGGGSAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLTGGGS 44 PD-1MQIPQAPWPVVWAVLQLGWRPGWFLDSPDPWNPPTFFPALLVVTEGDNATFTCSFSNTSESFVLNWYRMSPSNQTDKLAAFPEDRSQPGQDCRFRVTQLPNGRDFHMSVVRARRNDSGTYLCGAISLAPKAQIKESLRAELRVTERRAEVPTAHPSPSPRPAGQFQTLVVGVVGGLLGSLVLLVWVLAVICSRAARGTIGARRTGQPLKEDPSAVPVFSVDYGELDFQWREKTPEPPVPCVPEQTEYATIVFPSGMGTSSPARRGSADGPRSAQPLRPED GHCSWPL 45PD-L-1 MRIFAVFIFMTYWHLLNAFTVTVPKDLYVVEYGSNMTIECKFPVEKQLDLAALIVYWEMEDKNIIQFVHGEEDLKVQHSSYRQRARLLKDQLSLGNAALQITDVKLQDAGVYRCMISYGGADYKRITVKVNAPYNKINQRILVVDPVTSEHELTCQAEGYPKAEVIWTSSDHQVLSGKTTTTNSKREEKLFNVTSTLRINTTTNEIFYCTFRRLDPEENHTAELVTPELPLAHPPNERTHLVTLGAILLC

LGVALTFIFR LRKGRMMDVKKCGIQDTNSK

KQSDTHLEET 46 CTLA-4 MACLGFQRHKAQLNLATRTWPCTLLFFLLFIPVFCKAMHVAQPAWLASSRGIASFVCEYASPGKATEVRVTVLRQADSQVTEVCAATYMMGNELTFLDDSICTGTSSGNQVNLTIQGLRAMDTGLYICKVELMYPPPYY LGIGNGTQIY

VIDPEPCPDS

DFLLWILAAVSSGLFFYSFL LYAVSLSKML

KKRSPLTTGVYVKMPPTEPE

CEKQFQPYFI

PIN 47 LAG3 MWEAQFLGLLFLQPLWVAPVKPLQPGAEVPVVWAQEGAPAQLPCSPTIPLQDLSLLRRAGVTWQHQPDSGPPAAAPGHPLAPGPHPAAPSSWGPRPRRYTVLSVGPGGLRSGRLPLQPRVQLDERGRQRGDFSLWLRPAR

RADAGEYRAAVHLRDRALSCRLRLRLGQASMTASPPGSLR

ASDWVILNCSFSRPDRPASVHWFRNRGQGRVPVRESPHHHLAESFLFLPQVSPMDSGPWGCILTYRDGFNVSIMYNLTVLGLEPPTPLTVYAGAGSRVGLPCRLPAGVGTRSFLTAKWTPPGGGPDLLVTGDNGDFTLRL

EDVSQAQAGT

YTCHIHLQEQ

QLNATVTLAI

ITVTPKSFGS

PGSLGKLLCEVTPVSGQERFVWSSLDTPSQRSFSGPWLEAQEAQLLSQPWQCQLYQ GERLLGAAVYFTEL

SSPGAQRSGR

APGALPAGHL

LLFLILGVLS

LLLLVTGAFG

FHLWRRQWRPRRFSALEQGI

HPPQAQSKIE

ELEQEPEPEP EPEPEPEPEP

EPEQL 48 TIM3 MFSHLPFDCVLLLLLLLLTRSSEVEYRAEVGQNAYLPCFYTPAAPGNLVPVCWGKGACPVFECGNVVLRTDERDVNYWTSRYWLNGDFRKGDVSLTIENVTLADSGIYCCRIQIPGIMNDEKFNLKLVIKPAKVTPAPTR

QRDFTAAFPR

MLTTRGHGPA

ETQTLGSLPD

INLTQISTLA

NELRDSRLANDLRDSGATIRGIYIGAGICAGLALALIFGALIFKWYSHSKEKIQNLSLISLANLPPSGLANAVAEGIRSEENIYTIEENVYEVEEPNEYYCYVSSRQQPSQP LGCRFAMP 49B7-H3 MLRRRGSPGMGVHVGAALGALWFCLTGALEVQVPEDPVVALVGTDATLCC

SFSPEPGFSLQLNLIWQLT

DTKQLVHSFA

EGQDQGSAYA

NRTALFPDLLAQGNASLRLQRVRVADEGSFCFVSIRDFGSAAVSLQVAA

PYSKPSMTLE

PNKDLRPGDT

VTITCSSYQG

YPEAEVFWQD

GQGVPLTGNVTTSQMANEQGLFDVHSILRWLGANGTYSCLVRNPVLQQD

AHSSVTITPQRSPTGAVEVQVPEDPVVALVGTDATLRCSF

SPEPGFSLAQLNLIWQLTDTKQLVHSFTEGRDQGSAYANRTALFPDLLAQ

GNASLRLQRV

RVADEGSFTC

FVSIRDFGSA

AVSLQVAAPY

SKPSMTLEPNKDLRPGDTVTITCSSYRGYPEAEVFWQDGQGVPLTGNVTT

SQMANEQGLFDVHSVLRVVLGANGTYSCLVRNPVLQQDAH

GSVTITGQPMTFPPEALWVTVGLSVCLIALLVALAFVCWRKIKQSCEEEN

AGAEDQDGEGEGSKTALQPLKHSDSKEDDGQEIA 50 B7-H4MASLGQILFWSIISIIIILAGAIALIIGFGISAFSMPEVNVDYNASSETLRCEAPRWFPQPTVVWASQVDQGANFSEVSNTSFELNSENVTMKVVSVLYN

VTINNTYSCM IENDIAKATGDIKVTESEIKRRSHLQLLNS

KASLCVSSFFAISWALLPLSPYLMLK 51 TNF-alphaGPQREEFPRDLSLISPLAQAVRSSSRTPSDKPVAHVVANPQAEGQLQWLNRRANAL extracellularLANGVELRDNQLVVPSEGLYLIYSQVLFKGQGCPSTHVLLTHTISRIAVSYQTKVN domainLLSAIKSPCQRETPEGAEAKPWYEPIYLGGVFQLEKGDRLSAEINRPDYLDFAESG QVYFGIIAL 52LIGHT LQLHWRLGEMVTRLPDGPAGSWEQLIQERRSHEVNPAAHLTGANSSLTGSGGPLLWextracellular ETQLGLAFLRGLSYHDGALVVTKAGYYYIYSKVQLGGVGCPLGLASTITHGLYKRTdomain PRYPEELELLVSQQSPCGRATSSSRVWWDSSFLGGVVHLEAGEKVVVRVLDERLVRLRDGTRSYFGAFMV 53 LT-alpha LPGVGLTPSAAQTARQHPKMHLAHSTLKPAAHLIGDPSKQNSLLWRANTDRAFLQD extracellular GFSLSNNSLLVPTSGIYFVYSQVVFSGKAYSPKATSSPLYLAHEVQLFSSQYPFHV domainPLLSSQKMVYPGLQEPWLHSMYHGAAFQLTQGDQLSTHTDGIPHLVLSPSTVFFGA FAL 54 LT-beta QDQGGLVTETADPGAQAQQGLGFQKLPEEEPETDLSPGLPAAHLIGAPLKGQGLGW extracellular ETTKEQAFLTSGTQFSDAEGLALPQDGLYYLYCLVGYRGRAPPGGGDPQGRSVTLR domainSSLYRAGGAYGPGTPELLLEGAETVTPVLDPARRQGYGPLWYTSVGFGGLVQLRRGERVYVNISHPDMVDFARGKTFFGAVMVG 55 BTLAKESCDVQLYIKRQSEHSILAGDPFELECPVKYCANRPHVTWCKLNGTTCVKLEDRQ extracellular TSWKEEKNISFFILHFEPVLPNDNGSYRCSANFQSNLIESHSTTLYVTDVKSASER domainPSKDEMASRPWLLYR 56 CD160 INITSSASQEGTRLNLICTVWHKKEEAEGFVVFLCKDRSGDCSPETSLKQLRLKRD extracellular PGIDGVGEISSQLMFTISQVTPLHSGTYQCCARSQKSGIRLQGHFFSILFTETGNY domainTVTGLKQRQHLEFSHNEGTLS 57 CD40LMQKGDQNPQIAAHVISEASSKTTSVLQWAEKGYYTMSNNLVTLENGKQLTVKRQGL extracellular YYIYAQVTFCSNREASSQAPFIASLCLKSPGRFERILLRAANTHSSAKPCGQQSIH domainLGGVFELQPGASVFVNVTDPSQVSHGTGFTSFGLLKL 58 FasLQIGHPSPPPEKKELRKVAHLTGKSNSRSMPLEWEDTYGIVLLSGVKYKKGGLVINE extracellular TGLYFVYSKVYFRGQSCNNLPLSHKVYMRNSKYPQDLVMMEGKMMSYCTTGQMWAR domainSSYLGAVFNLTSADHLYVNVSELSLVNFEESQTFFGLYKL 59 CD30LFPQDRPFEDTCHGNPSHYYDKAVRRCCYRCPMGLFPTQQCPQRPTDCRKQCEPDYY extracellular LDEADRCTACVTCSRDDLVEKTPCAWNSSRVCECRPGMFCSTSAVNSCARCFFHSV domainCPAGMIVKFPGTAQKNTVCEPASPGVSPACASPENCKEPSSGTIPQAKPTPVSPATSSASTMPVRGGTRLAQEAASKLTRAPDSPSSVGRPSSDPGLSPTQPCPEGSGDCRKQCEPDYYLDEAGRCTACVSCSRDDLVEKTPCAWNSSRTCECRPGMICATSATNSCARCVPYPICAAETVTKPQDMAEKDTTFEAPPLGTQPDCNPTPENGEAPASTSPTQSLLVDSQASKTLPIPTSAPVALSSTGKPVLDAGPVLFWVILVLVVVVGSSAFLLCHRRACRKRIRQKLHLCYPVQTSQPKLELVDSRPRRSSTQLRSGASVTEPVAEERGLMSQPLMETCHSVGAAYLESLPLQDASPAGGPSSPRDLPEPRVSTEHTNNKIEKIYIMKADTVIVGTVKAELPEGRGLAGPAEPELEEELEADHTPHYPEQETEPPLGSCSDVMLSVEEEGKEDPLPTAASGK 60 4-1BBLACPWAVSGARASPGSAASPRLREGPELSPDDPAGLLDLRQGMFAQLVAQNVLLIDG extracellular PLSWYSDPGLAGVSLTGGLSYKEDTKELVVAKAGVYYVFFQLELRRVVAGEGSGSV domainSLALHLQPLRSAAGAAALALTVDLPPASSEARNSAFGFQGRLLHLSAGQRLGVHLHTEARARHAWQLTQGATVLGLFRVTPEIPAGLPSPRSE 61 CD27LATPAPKSCPERHYWAQGKLCCQMCEPGTFLVKDCDQHRKAAQCDPCIPGVSFSPDH extracellular HTRPHCESCRHCNSGLLVRNCTITANAECACRNGWQCRDKECTECDPLPNPSLTAR domainSSQALSPHPQPTHLPYVSEMLEARTAGHMQTLADFRQLPARTLSTHWPPQRSLCSS DFIR 62 OX40LLHCVGDTYPSNDRCCHECRPGNGMVSRCSRSQNTVCRPCGPGFYNDVVSSKPCKPC extracellular TWCNLRSGSERKQLCTATQDTVCRCRAGTQPLDSYKPGVDCAPCPPGHFSPGDNQA domainCKPWTNCTLAGKHTLQPASNSSDAICEDRDPPATQPQETQGPPARPITVQPTEAWPRYSQGPSTRPVEVPGGRA 63 TWEAK SAPKGRKTRARRAIAAHYEVHPRPGQDGAQAGVDGTVSGWEEARINSSSPLRYNRQ extracellular IGEFIVTRAGLYYLYCQVHFDEGKAVYLKLDLLVDGVLALRCLEEFSATAASSLGP domainQLRLCQVSGLLALRPGSSLRIRTLPWAHLKAAPFLTYFGLFQVH 64 APRIL  AVLTQKQKKQHSVLHLVPINATSKDDSDVTEVMWQPALRRGRGLQAQGYGVRIQDA extracellularGVYLLYSQVLFQDVTFTMGVVWSREGQGRQETLFRCIRSMPSHPDRAYNSCYSAGV domainFHLHQGDILSVIIPRARAKLNLSPHGTFLGFVKL 65 BAFF  AVQGPEETVTQDCLQLIADSETPTIQKGSYTFVPWLLSFKRGSALEEKENKILVKE extracellularTGYFFIYGQVLYTDKTYAMGHLIQRKKVHVFGDELSLVTLFRCIQNMPETLPNNSC domainYSAGIAKLEEGDELQLAIPRENAQISLDGDVTFFGALKLL 66 RANKL YFRAQMDPNRISEDGTHCIYRILRLHENADFQDTTLESQDTKLIPDSCRRIKQAFQ extracellular GAVQKELQHIVGSQHIRAEKAMVDGSWLDLAKRSKLEAQPFAHLTINATDIPSGSH domainKVSLSSWYHDRGWAKISNMTFSNGKLIVNQDGFYYLYANICFRHHETSGDLATEYLQLMVYVTKTSIKIPSSHTLMKGGSTKYWSGNSEFHFYSINVGGFFKLRSGEEISIEVSNPSLLDPDQDATYFGAFKVRDID 67 TRAIL  TNELKQMQDKYSKSGIACFLKEDDSYWDPNDEESMNSPCWQVKWQLRQLVRKMILR extracellularTSEETISTVQEKQQNISPLVRERGPQRVAAHITGTRGRSNTLSSPNSKNEKALGRK domainINSWESSRSGHSFLSNLHLRNGELVIHEKGFYYIYSQTYFRFQEEIKENTKNDKQMVQYIYKYTSYPDPILLMKSARNSCWSKDAEYGLYSIYQGGIFELKENDRIFVSVTNEHLIDMDHEASFFGAFLVG 68 EDA1 ELRSELRRERGAESRLGGSGTPGTSGTLSSLGGLDPDSPITSHLGQPSPKQQPLEP extracellular GEAALHSDSQDGHQMALLNFFFPDEKPYSEEESRRVRRNKRSKSNEGADGPVKNKK domainKGKKAGPPGPNGPPGPPGPPGPQGPPGIPGIPGIPGTTVMGPPGPPGPPGPQGPPGLQGPSGAADKAGTRENQPAVVHLQGQGSAIQVKNDLSGGVLNDWSRITMNPKVFKLHPRSGELEVLVDGTYFIYSQVEVYYINFTDFASYEVVVDEKPFLQCTRSIETGKTNYNTCYTAGVCLLKARQKIAVKMVHADISINMSKHTTFFGAIRLGEAPAS 69 EDA2 

extracellular  ELRSELRRERGAESRLGGSGTPGTSGTLSSLGGLDPDSPITSHLGQPSPKQQPLEPdomain GEAALHSDSQDGHQGHQ 70 GITRL QLETAKEPCMAKFGPLPSKWQMASSEPPCVNKVSDWKLEILQNGLYLIYGQVAPNA extracellular NYNDVAPFEVRLYKNKDMIQTLTNKSKIQNVGGTYELHVGDTIDLIFNSEHQVLKN domainNTYWGIILLANPQFIS 71 CD80 (B7-1) VIHVTKEVKEVATLSCGHNVSVEELAQTRIYWQKEKKMVLTMMSGDMNIWPEYKNR extracellular TIFDITNNLSIVILALRPSDEGTYECVVLKYEKDAFKREHLAEVTLSVKADFPTPS domainISDFEIPTSNIRRIICSTSGGFPEPHLSWLENGEELNAINTTVSQDPETELYAVSSKLDFNMTTNHSFMCLIKYGHLRVNQTFNWNTTKQEHFPDN 72 CD86 (B7-2) APLKIQAYFNETADLPCQFANSQNQSLSELVVFWQDQENLVLNEVYLGKEKFDSVH extracellular SKYMGRTSFDSDSWTLRLHNLQIKDKGLYQCIIHHKKPTGMIRIHQMNSELSVLAN domainFSQPEIVPISNITENVYINLTCSSIHGYPEPKKMSVLLRTKNSTIEYDGVMQKSQDNVTELYDVSISLSVSFPDVTSNMTIFCILETDKTRLLSSPFSIELEDPQPPPDHIP 73 ICOSLG DTQEKEVRAMVGSDVELSCACPEGSRFDLNDVYVYWQTSESKTVVTYHIPQNSSLE extracellular NVDSRYRNRALMSPAGMLRGDFSLRLFNVTPQDEQKFHCLVLSQSLGFQEVLSVEV domainTLHVAANFSVPVVSAPHSPSQDELTFTCTSINGYPRPNVYWINKTDNSLLDQALQNDTVFLNMRGLYDVVSVLRIARTPSVNIGCCIENVLLQQNLTVGSQTGNDIGERDKI TENPVSTGEKNAAT74 MICA  EPHSLRYNLTVLSWDGSVQSGFLTEVHLDGQPFLRCDRQKCRAKPQGQWAEDVLGNextracellular  KTWDRETRDLTGNGKDLRMTLAHIKDQKEGLHSLQEIRVCEIHEDNSTRSSQHFYYdomain DGELFLSQNLETKEWTMPQSSRAQTLAMNVRNFLKEDAMKTKTHYHAMHADCLQELRRYLKSGVVLRRTVPPMVNVTRSEASEGNITVTCRASGFYPWNITLSWRQDGVSLSHDTQQWGDVLPDGNGTYQTWVATRICQGEEQRFTCYMEHSGNHSTHPVPSGKVLVL QSHW 75 MICB AEPHSLRYNLMVLSQDESVQSGFLAEGHLDGQPFLRYDRQKRRAKPQGQWAEDVLG extracellular AKTWDTETEDLTENGQDLRRTLTHIKDQKGGLHSLQEIRVCEIHEDSSTRGSRHFY domainYDGELFLSQNLETQESTVPQSSRAQTLAMNVTNFWKEDAMKTKTHYRAMQADCLQKLQRYLKSGVAIRRTVPPMVNVTCSEVSEGNITVTCRASSFYPRNITLTWRQDGVSLSHNTQQWGDVLPDGNGTYQTWVATRIRQGEEQRFTCYMEHSGNHGTHPVPSGKVLV LQSQRTD 76ULBP1 GWVDTHCLCYDFIITPKSRPEPQWCEVQGLVDERPFLHYDCVNHKAKAFASLGKKVextracellular NVTKTWEEQTETLRDVVDFLKGQLLDIQVENLIPIEPLTLQARMSCEHEAHGHGRGdomain SWQFLFNGQKFLLFDSNNRKWTALHPGAKKMTEKWEKNRDVTMFFQKISLGDCKMWLEEFLMYWEQMLDPTKPPSLAPG 77 ULBP2GRADPHSLCYDITVIPKFRPGPRWCAVQGQVDEKTFLHYDCGNKTVTPVSPLGKKL extracellularNVTTAWKAQNPVLREVVDILTEQLRDIQLENYTPKEPLTLQARMSCEQKAEGHSSG domainSWQFSFDGQIFLLFDSEKRMWTTVHPGARKMKEKVVENDKWAMSFHYFSMGDCIGWLEDFLMGMDSTLEPSAGAPLAMS 78 ULBP3DAHSLWYNFTIIHLPRHGQQWCEVQSQVDQKNFLSYDCGSDKVLSMGHLEEQLYAT extracellularDAWGKQLEMLREVGQRLRLELADTELEDFTPSGPLTLQVRMSCECEADGYIRGSWQ domainFSFDGRKFLLFDSNNRKWTVVHAGARRMKEKWEKDSGLTTFFKMVSMRDCKSWLRDFLMHRKKRLEPTAPPTMAPG 79 ULBP4HSLCFNFTIKSLSRPGQPWCEAQVFLNKNLFLQYNSDNNMVKPLGLLGKKVYATST extracellularWGELTQTLGEVGRDLRMLLCDIKPQIKTSDPSTLQVEMFCQREAERCTGASWQFAT domainNGEKSLLFDAMNMTWTVINHEASKIKETWKKDRGLEKYFRKLSKGDCDHWLREFLGHWEAMPEPTVSPVNASDIHWSSSSLPD 80 ULBP5,GLADPHSLCYDITVIPKFRPGPRWCAVQGQVDEKTFLHYDCGSKTVTPVSPLGKKL isoform 1NVTTAWKAQNPVLREVVDILTEQLLDIQLENYIPKEPLTLQARMSCEQKAEGHGSG extracellularSWQLSFDGQIFLLFDSENRMWTTVHPGARKMKEKWENDKDMTMSFHYISMGDCTGW domainLEDFLMGMDSTLEPSAGAPPTMSSG 81 ULBP5,GLADPHSLCYDITVIPKFRPGPRWCAVQGQVDEKTFLHYDCGSKTVTPVSPLGKKL isoform 2NVTTAWKAQNPVLREVVDILTEQLLDIQLENYIPKEPLTLQARMSCEQKAEGHGSG extracellularSWQLSFDGQIFLLFDSENRMWTTVHPGARKMKEKWENDKDMTMSFHYISMGDCTGW domainLEDFLMGMDSTLEPSAGGTV 82 ULBP6RRDDPHSLCYDITVIPKFRPGPRWCAVQGQVDEKTFLHYDCGNKTVTPVSPLGKKL extracellularNVTMAWKAQNPVLREVVDILTEQLLDIQLENYTPKEPLTLQARMSCEQKAEGHSSG domainSWQFSIDGQTFLLFDSEKRMWTTVHPGARKMKEKWENDKDVAMSFHYISMGDCIGWLEDFLMGMDSTLEPSAGAPLAMSSG 83 SLAMF1ASYGTGGRMMNCPKILRQLGSKVLLPLTYERINKSMNKSIHIVVTMAKSLENSVEN extracellularKIVSLDPSEAGPPRYLGDRYKFYLENLTLGIRESRKEDEGWYLMTLEKNVSVQRFC domainLQLRLYEQVSTPEIKVLNKTQENGTCTLILGCTVEKGDHVAYSWSEKAGTHPLNPANSSHLLSLTLGPQHADNIYICTVSNPISNNSQTFSPWPGCRTDPSETKP 84 SLAMF2QGHLVHMTVVSGSNVTLNISESLPENYKQLTWFYTFDQKIVEWDSRKSKYFESKFK extracellularGRVRLDPQSGALYISKVQKEDNSTYIMRVLKKTGNEQEWKIKLQVLDPVPKPVIKI domainEKIEDMDDNCYLKLSCVIPGESVNYTWYGDKRPFPKELQNSVLETTLMPHNYSRCYTCQVSNSVSSKNGTVCLSPPCTLARS 85 SLAMF3KDSAPTVVSGILGGSVTLPLNISVDTEIENVIWIGPKNALAFARPKENVTIMVKSY extracellularLGRLDITKWSYSLCISNLTLNDAGSYKAQINQRNFEVTTEEEFTLFVYEQLQEPQV domainTMKSVKVSENFSCNITLMCSVKGAEKSVLYSWTPREPHASESNGGSILTVSRTPCDPDLPYICTAQNPVSQRSSLPVHVGQFCTDPGASRGGTTGETVVGVLGEPVTLPLALPACRDTEKVVWLFNTSIISKEREEAATADPLIKSRDPYKNRVWVSSQDCSLKISQLKIEDAGPYHAYVCSEASSVTSMTHVTLLIYRRLRKPKITWSLRHSEDGICRISLTCSVEDGGNTVMYTWTPLQKEAWSQGESHLNVSWRSSENHPNLTCTASNPVSRSSHQ FLSENICSGPERNTK86 SLAMF4 CQGSADHVVSISGVPLQLQPNSIQTKVDSIAWKKLLPSQNGFHHILKWENGSLPSNextracellular TSNDRFSFIVKNLSLLIKAAQQQDSGLYCLEVTSISGKVQTATFQVFVFESLLPDKdomain VEKPRLQGQGKILDRGRCQVALSCLVSRDGNVSYAWYRGSKLIQTAGNLTYLDEEVDINGTHTYTCNVSNPVSWESHTLNLTQDCQNAHQEFRFWP 87 SLAMF5KDSEIFTVNGILGESVTFPVNIQEPRQVKIIAWTSKTSVAYVTPGDSETAPVVTVT extracellularHRNYYERIHALGPNYNLVISDLRMEDAGDYKADINTQADPYTTTKRYNLQIYRRLG domainKPKITQSLMASVNSTCNVTLTCSVEKEEKNVTYNWSPLGEEGNVLQIFQTPEDQELTYTCTAQNPVSNNSDSISARQLCADIAMGFRTHHTG 88 SLAMF6QSSLTPLMVNGILGESVTLPLEFPAGEKVNFITWLFNETSLAFIVPHETKSPEIHV extracellularTNPKQGKRLNFTQSYSLQLSNLKMEDTCSYRAQISTKTSAKLSSYTLRILRQLRNI domainQVTNHSQLFQNMTCELHLTCSVEDADDNVSFRWEALGNTLSSQPNLTVSWDPRISSEQDYTCIAENAVSNLSFSVSAQKLCEDVKIQYTDTKM 89 SLAMF7SGPVKELVGSVGGAVTFPLKSKVKQVDSIVWTFNTTPLVTIQPEGGTIIVTQNRNR extracellularERVDFPDGGYSLKLSKLKKNDSGIYYVGIYSSSLQQPSTQEYVLHVYEHLSKPKVT domainMGLQSNKNGTCVTNLTCCMEHGEEDVIYTWKALGQAANESHNGSILPISWRWGESDMTFICVARNPVSRNFSSPILARKLCEGAADDPDSSM 90 human alpha1MFSFVDLRLLLLLAATALLTHGQEEGQVEGQDEDIPPITCVQNGLRYHDRDVWKPE chainPCRICVCDNGKVLCDDVICDETKNCPGAEVPEGECCPVCPDGSESPTDQETTGVEG precurson ofPKGDTGPRGPRGPAGPPGRDGIPGQPGLPGPPGPPGPPGPPGLGGNFAPQLSYGYD type I collagenEKSTGGISVPGPMGPSGPRGLPGPPGAPGPQGFQGPPGEPGEPGASGPMGPRGPPG (NCBIPPGKNGDDGEAGKPGRPGERGPPGPQGARGLPGTAGLPGMKGHRGFSGLDGAKGDA ReferenceGPAGPKGEPGSPGENGAPGQMGPRGLPGERGRPGAPGPAGARGNDGATGAAGPPGP Sequence:TGPAGPPGFPGAVGAKGEAGPQGPRGSEGPQGVRGEPGPPGPAGAAGPAGNPGADG NP_000079.2)QPGAKGANGAPGIAGAPGFPGARGPSGPQGPGGPPGPKGNSGEPGAPGSKGDTGAKGEPGPVGVQGPPGPAGEEGKRGARGEPGPTGLPGPPGERGGPGSRGFPGADGVAGPKGPAGERGSPGPAGPKGSPGEAGRPGEAGLPGAKGLTGSPGSPGPDGKTGPPGPAGQDGRPGPPGPPGARGQAGVMGFPGPKGAAGEPGKAGERGVPGPPGAVGPAGKDGEAGAQGPPGPAGPAGERGEQGPAGSPGFQGLPGPAGPPGEAGKPGEQGVPGDLGAPGPSGARGERGFPGERGVQGPPGPAGPRGANGAPGNDGAKGDAGAPGAPGSQGAPGLQGMPGERGAAGLPGPKGDRGDAGPKGADGSPGKDGVRGLTGPIGPPGPAGAPGDKGESGPSGPAGPTGARGAPGDRGEPGPPGPAGFAGPPGADGQPGAKGEPGDAGAKGDAGPPGPAGPAGPPGPIGNVGAPGAKGARGSAGPPGATGFPGAAGRVGPPGPSGNAGPPGPPGPAGKEGGKGPRGETGPAGRPGEVGPPGPPGPAGEKGSPGADGPAGAPGTPGPQGIAGQRGVVGLPGQRGERGFPGLPGPSGEPGKQGPSGASGERGPPGPMGPPGLAGPPGESGREGAPGAEGSPGRDGSPGAKGDRGETGPAGPPGAPGAPGAPGPVGPAGKSGDRGETGPAGPAGPVGPVGARGPAGPQGPRGDKGETGEQGDRGIKGHRGFSGLQGPPGPPGSPGEQGPSGASGPAGPRGPPGSAGAPGKDGLNGLPGPIGPPGPRGRTGDAGPVGPPGPPGPPGPPGPPSAGFDFSFLPQPPQEKAHDGGRYYRADDANVVRDRDLEVDTTLKSLSQQIENIRSPEGSRKNPARTCRDLKMCHSDWKSGEYWIDPNQGCNLDAIKVFCNMETGETCVYPTQPSVAQKNWYISKNPKDKRHVWFGESMTDGFQFEYGGQGSDPADVAIQLTFLRLMSTEASQNITYHCKNSVAYMDQQTGNLKKALLLQGSNEIEIRAEGNSRFTYSVTVDGCTSHTGAWGKTVIEYKTTKTSRLPIIDVAPLDVGAPDQEFGF DVGPVCFL 91human alpha2 MLSFVDTRTLLLLAVTLCLATCQSLQEETVRKGPAGDRGPRGERGPPGPPGRDGEDchain GPTGPPGPPGPPGPPGLGGNFAAQYDGKGVGLGPGPMGLMGPRGPPGAAGAPGPQGprecurson of FQGPAGEPGEPGQTGPAGARGPAGPPGKAGEDGHPGKPGRPGERGVVGPQGARGFPtype I collagen GTPGLPGFKGIRGHNGLDGLKGQPGAPGVKGEPGAPGENGTPGQTGARGLPGERGR(NCBI VGAPGPAGARGSDGSVGPVGPAGPIGSAGPPGFPGAPGPKGEIGAVGNAGPAGPAG ReferencePRGEVGLPGLSGPVGPPGNPGANGLTGAKGAAGLPGVAGAPGLPGPRGIPGPVGAA Sequence:GATGARGLVGEPGPAGSKGESGNKGEPGSAGPQGPPGPSGEEGKRGPNGEAGSAGP NP_000080.2)PGPPGLRGSPGSRGLPGADGRAGVMGPPGSRGASGPAGVRGPNGDAGRPGEPGLMGPRGLPGSPGNIGPAGKEGPVGLPGIDGRPGPIGPAGARGEPGNIGFPGPKGPTGDPGKNGDKGHAGLAGARGAPGPDGNNGAQGPPGPQGVQGGKGEQGPPGPPGFQGLPGPSGPAGEVGKPGERGLHGEFGLPGPAGPRGERGPPGESGAAGPTGPIGSRGPSGPPGPDGNKGEPGVVGAVGTAGPSGPSGLPGERGAAGIPGGKGEKGEPGLRGEIGNPGRDGARGAPGAVGAPGPAGATGDRGEAGAAGPAGPAGPRGSPGERGEVGPAGPNGFAGPAGAAGQPGAKGERGAKGPKGENGVVGPTGPVGAAGPAGPNGPPGPAGSRGDGGPPGMTGFPGAAGRTGPPGPSGISGPPGPPGPAGKEGLRGPRGDQGPVGRTGEVGAVGPPGFAGEKGPSGEAGTAGPPGTPGPQGLLGAPGILGLPGSRGERGLPGVAGAVGEPGPLGIAGPPGARGPPGAVGSPGVNGAPGEAGRDGNPGNDGPPGRDGQPGHKGERGYPGNIGPVGAAGAPGPHGPVGPAGKHGNRGETGPSGPVGPAGAVGPRGPSGPQGIRGDKGEPGEKGPRCLPGLKGHNGLQGLPGIAGHHGDQGAPGSVGPAGPRGPAGPSGPAGKDGRTGHPGTVGPAGIRGPQGHQGPAGPPGPPGPPGPPGVSGGGYDFGYDGDFYRADQPRSAPSLRPKDYEVDATLKSLNNQIETLLTPEGSRKNPARTCRDLRLSHPEWSSGYYWIDPNQGCTMDAIKVYCDFSTGETCIRAQPENIPAKNWYRSSKDKKHVWLGETINAGSQFEYNVEGVTSKEMATQLAFMRLLANYASQNITYHCKNSIAYMDEETGNLKKAVILQGSNDVELVAEGNSRFTYTVLVDGCSKKTNEWGKTIIEYKTNKPSRLPFLDIAPLDIGGADQEFFVDIGPVCFK 92 human alpha1MQGPEGPQGPPGQKGDTGEPGLPGTKGTRGPPGASGYPGNPGLPGIPGQDGPPGPP chain of typeGIPGCNGTKGERGPLGPPGLPGFAGNPGPPGLPGMKGDPGEILGHVPGMLLKGERG IV collagenFPGIPGTPGPPGLPGLQGPVGPPGFTGPPGPPGPPGPPGEKGQMGLSFQGPKGDKG (NCBIDQGVSGPPGVPGQAQVQEKGDFATKGEKGQKGEPGFQGMPGVGEKGEPGKPGPRGK ReferencePGKDGDKGEKGSPGFPGEPGYPGLIGRQGPQGEKGEAGPPGPPGIVIGTGPLGEKG Sequence:ERGYPGTPGPRGEPGPKGFPGLPGQPGPPGLPVPGQAGAPGFPGERGEKGDRGFPG XP_011519350.1)TSLPGPSGRDGLPGPPGSPGPPGQPGYTNGIVECQPGPPGDQGPPGIPGQPGFIGEIGEKGQKGESCLICDIDGYRGPPGPQGPPGEIGFPGQPGAKGDRGLPGRDGVAGVPGPQGTPGLIGQPGAKGEPGEFYFDLRLKGDKGDPGFPGQPGMPGRAGSPGRDGHPGLPGPKGSPGSVGLKGERGPPGGVGFPGSRGDTGPPGPPGYGPAGPIGDKGQAGFPGGPGSPGLPGPKGEPGKIVPLPGPPGAEGLPGSPGFPGPQGDRGFPGTPGRPGLPGEKGAVGQPGIGFPGPPGPKGVDGLPGDMGPPGTPGRPGFNGLPGNPGVQGQKGEPGVGLPGLKGLPGLPGIPGTPGEKGSIGVPGVPGEHGAIGPPGLQGIRGEPGPPGLPGSVGSPGVPGIGPPGARGPPGGQGPPGLSGPPGIKGEKGFPGFPGLDMPGPKGDKGAQGLPGITGQSGLPGLPGQQGAPGIPGFPGSKGEMGVMGTPGQPGSPGPVGAPGLPGEKGDHGFPGSSGPRGDPGLKGDKGDVGLPGKPGSMDKVDMGSMKGQKGDQGEKGQIGPIGEKGSRGDPGTPGVPGKDGQAGQPGQPGPKGDPGISGTPGAPGLPGPKGSVGGMGLPGTPGEKGVPGIPGPQGSPGLPGDKGAKGEKGQAGPPGIGIPGLRGEKGDQGIAGFPGSPGEKGEKGSIGIPGMPGSPGLKGSPGSVGYPGSPGLPGEKGDKGLPGLDGIPGVKGEAGLPGTPGPTGPAGQKGEPGSDGIPGSAGEKGEPGLPGRGFPGFPGAKGDKGSKGEVGFPGLAGSPGIPGSKGEQGFMGPPGPQGQPGLPGSPGHATEGPKGDRGPQGQPGLPGLPGPMGPPGLPGIDGVKGDKGNPGWPGAPGVPGPKGDPGFQGMPGIGGSPGITGSKGDMGPPGVPGFQGPKGLPGLQGIKGDQGDQGVPGAKGLPGPPGPPGPYDIIKGEPGLPGPEGPPGLKGLQGLPGPKGQQGVTGLVGIPGPPGIPGFDGAPGQKGEMGPAGPTGPRGFPGPPGPDGLPGSMGPPGTPSVDHGFLVTRHSQTIDDPQCPSGTKILYHGYSLLYVQGNERAHGQDLGTAGSCLRKFSTMPFLFCNINNVCNFASRNDYSYWLSTPEPMPMSMAPITGENIRPFISRCAVCEAPAMVMAVHSQTIQIPPCPSGWSSLWIGYSFVMHTSAGAEGSGQALASPGSCLEEFRSAPFIECHGRGTCNYYANAYSFWLATIERSEMFKKPTPSTLKAGELRTHVSRCQVCMRRT 93MGRDQRAVAGPALRRWLLLGTVTVGFLAQSVLAGVKKFDVPCGGRDCSGGCQCYPEKGGRGQPGPVGPQGYNGPPGLQGFPGLQGRKGDKGERGAPGVTGPKGDVGARGVSGFPGADGIPGHPGQGGPRGRPGYDGCNGTQGDSGPQGPPGSEGFTGPPGPQGPKGQKGEPYALPKEERDRYRGEPGEPGLVGFQGPPGRPGHVGQMGPVGAPGRPGPPGPPGPKGQQGNRGLGFYGVKGEKGDVGQPGPNGIPSDTLHPIIAPTGVTFHPDQYKGEKGSEGEPGIRGISLKGEEGIMGFPGLRGYPGLSGEKGSPGQKGSRGLDGYQGPDGPRGPKGEAGDPGPPGLPAYSPHPSLAKGARGDPGFPGAQGEPGSQGEPGDPGLPGPPGLSIGDGDQRRGLPGEMGPKGFIGDPGIPALYGGPPGPDGKRGPPGPPGLPGPPGPDGFLFGLKGAKGRAGFPGLPGSPGARGPKGWKGDAGECRCTEGDEAIKGLPGLPGPKGFAGINGEPGRKGDRGDPGQHGLPGFPGLKGVPGNIGAPGPKGAKGDSRTITTKGERGQPGVPGVPGMKGDDGSPGRDGLDGFPGLPGPPGDGIKGPPGDPGYPGIPGTKGTPGEMGPPGLGLPGLKGQRGFPGDAGLPGPPGFLGPPGPAGTPGQIDCDTDVKRAVGGDRQEAIQPGCIGGPKGLPGLPGPPGPTGAKGLRGIPGFAGADGGPGPRGLPGDAGREGFPGPPGFIGPRGSKGAVGLPGPDGSPGPIGLPGPDGPPGERGLPGEVLGAQPGPRGDAGVPGQPGLKGLPGDRGPPGFRGSQGMPGMPGLKGQPGLPGPSGQPGLYGPPGLHGFPGAPGQEGPLGLPGIPGREGLPGDRGDPGDTGAPGPVGMKGLSGDRGDAGFTGEQGHPGSPGFKGIDGMPGTPGLKGDRGSPGMDGFQGMPGLKGRPGFPGSKGEAGFFGIPGLKGLAGEPGFKGSRGDPGPPGPPPVILPGMKDIKGEKGDEGPMGLKGYLGAKGIQGMPGIPGLSGIPGLPGRPGHIKGVKGDIGVPGIPGLPGFPGVAGPPGITGFPGFIGSRGDKGAPGRAGLYGEIGATGDFGDIGDTINLPGRPGLKGERGTTGIPGLKGFFGEKGTEGDIGFPGITGVTGVQGPPGLKGQTGFPGLTGPPGSQGELGRIGLPGGKGDDGWPGAPGLPGFPGLRGIRGLHGLPGTKGFPGSPGSDIHGDPGFPGPPGERGDPGEANTLPGPVGVPGQKGDQGAPGERGPPGSPGLQGFPGITPPSNISGAPGDKGAPGIFGLKGYRGPPGPPGSAALPGSKGDTGNPGAPGTPGTKGWAGDSGPQGRPGVFGLPGEKGPRGEQGFMGNTGPTGAVGDRGPKGPKGDPGFPGAPGTVGAPGIAGIPQKIAVQPGTVGPQGRRGPPGAPGEMGPQGPPGEPGFRGAPGKAGPQGRGGVSAVPGFRGDEGPIGHQGPIGQEGAPGRPGSPGLPGMPGRSVSIGYLLVKHSQTDQEPMCPVGMNKLWSGYSLLYFEGQEKAHNQDLGLAGSCLARFSTMPFLYCNPGDVCYYASRNDKSYWLSTTAPLPMMPVAEDEIKPYISRCSVCEAPAIAIAVHSQDVSIPHCPAGWRSLWIGYSFLMHTAAGDEGGGQSLVSPGSCLEDFRATPFIECNGGRGTCHYYANKYSFWLTTIPEQSFQGSPSADTLKAGLIRTHISRCQVCMKNL 94 human alpha3MSARTAPRPQVLLLPLLLVLLAAAPAASKGCVCKDKGQCFCDGAKGEKGEKGFPGP chain of typePGSPGQKGFTGPEGLPGPQGPKGFPGLPGLTGSKGVRGISGLPGFSGSPGLPGTPG IV collagenNTGPYGLVGVPGCSGSKGEQGFPGLPGTLGYPGIPGAAGLKGQKGAPAKEEDIELD (NCBIAKGDPGLPGAPGPQGLPGPPGFPGPVGPPGPPGFFGFPGAMGPRGPKGHMGERVIG ReferenceHKGERGVKGLTGPPGPPGTVIVTLTGPDNRTDLKGEKGDKGAMGEPGPPGPSGLPG Sequence:ESYGSEKGAPGDPGLQGKPGKDGVPGFPGSEGVKGNRGFPGLMGEDGIKGQKGDIG NP_000082.2)PPGFRGPTEYYDTYQEKGDEGTPGPPGPRGARGPQGPSGPPGVPGSPGSSRPGLRGAPGWPGLKGSKGERGRPGKDAMGTPGSPGCAGSPGLPGSPGPPGPPGDIVFRKGPPGDHGLPGYLGSPGIPGVDGPKGEPGLLCTQCPYIPGPPGLPGLPGLHGVKGIPGRQGAAGLKGSPGSPGNTGLPGFPGFPGAQGDPGLKGEKGETLQPEGQVGVPGDPGLRGQPGRKGLDGIPGTPGVKGLPGPKGELALSGEKGDQGPPGDPGSPGSPGPAGPAGPPGYGPQGEPGLQGTQGVPGAPGPPGEAGPRGELSVSTPVPGPPGPPGPPGHPGPQGPPGIPGSLGKCGDPGLPGPDGEPGIPGIGFPGPPGPKGDQGFPGTKGSLGCPGKMGEPGLPGKPGLPGAKGEPAVAMPGGPGTPGFPGERGNSGEHGEIGLPGLPGLPGTPGNEGLDGPRGDPGQPGPPGEQGPPGRCIEGPRGAQGLPGLNGLKGQQGRRGKTGPKGDPGIPGLDRSGFPGETGSPGIPGHQGEMGPLGQRGYPGNPGILGPPGEDGVIGMMGFPGAIGPPGPPGNPGTPGQRGSPGIPGVKGQRGTPGAKGEQGDKGNPGPSEISHVIGDKGEPGLKGFAGNPGEKGNRGVPGMPGLKGLKGLPGPAGPPGPRGDLGSTGNPGEPGLRGIPGSMGNMGMPGSKGKRGTLGFPGRAGRPGLPGIHGLQGDKGEPGYSEGTRPGPPGPTGDPGLPGDMGKKGEMGQPGPPGHLGPAGPEGAPGSPGSPGLPGKPGPHGDLGFKGIKGLLGPPGIRGPPGLPGFPGSPGPMGIRGDQGRDGIPGPAGEKGETGLLRAPPGPRGNPGAQGAKGDRGAPGFPGLPGRKGAMGDAGPRGPTGIEGFPGPPGLPGAIIPGQTGNRGPPGSRGSPGAPGPPGPPGSHVIGIKGDKGSMGHPGPKGPPGTAGDMGPPGRLGAPGTPGLPGPRGDPGFQGFPGVKGEKGNPGFLGSIGPPGPIGPKGPPGVRGDPGTLKIISLPGSPGPPGTPGEPGMQGEPGPPGPPGNLGPCGPRGKPGKDGKPGTPGPAGEKGNKGSKGEPGPAGSDGLPGLKGKRGDSGSPATWTTRGFVFTRHSQTTAIPSCPEGTVPLYSGFSFLFVQGNQRAHGQDLGTLGSCLQRFTTMPFLFCNVNDVCNFASRNDYSYWLSTPALMPMNMAPITGRALEPYISRCTVCEGPAIAIAVHSQTTDIPPCPHGWISLWKGFSFIMFTSAGSEGTGQALASPGSCLEEFRASPFLECHGRGTCNYYSNSYSFWLASLNPERMFRKPIPSTVKAGELEKIISRCQVCMKKRH 95 human alpha4MWSLHIVLMRCSFRLTKSLATGPWSLILILFSVQYVYGSGKKYIGPCGGRDCSVCH chain of typeCVPEKGSRGPPGPPGPQGPIGPLGAPGPIGLSGEKGMRGDRGPPGAAGDKGDKGPT IV collagenGVPGFPGLDGIPGHPGPPGPRGKPGMSGHNGSRGDPGFPGGRGALGPGGPLGHPGE (NCBIKGEKGNSVFILGAVKGIQGDRGDPGLPGLPGSWGAGGPAGPTGYPGEPGLVGPPGQ ReferencePGRPGLKGNPGVGVKGQMGDPGEVGQQGSPGPTLLVEPPDFCLYKGEKGIKGIPGM Sequence:VGLPGPPGRKGESGIGAKGEKGIPGFPGPRGDPGSYGSPGFPGLKGELGLVGDPGL NP_000083.3)FGLIGPKGDPGNRGHPGPPGVLVTPPLPLKGPPGDPGFPGRYGETGDVGPPGPPGLLGRPGEACAGMIGPPGPQGFPGLPGLPGEAGIPGRPDSAPGKPGKPGSPGLPGAPGLQGLPGSSVIYCSVGNPGPQGIKGKVGPPGGRGPKGEKGNEGLCACEPGPMGPPGPPGLPGRQGSKGDLGLPGWLGTKGDPGPPGAEGPPGLPGKHGASGPPGNKGAKGDMVVSRVKGHKGERGPDGPPGFPGQPGSHGRDGHAGEKGDPGPPGDHEDATPGGKGFPGPLGPPGKAGPVGPPGLGFPGPPGERGHPGVPGHPGVRGPDGLKGQKGDTISCNVTYPGRHGPPGFDGPPGPKGFPGPQGAPGLSGSDGHKGRPGTPGTAEIPGPPGFRGDMGDPGFGGEKGSSPVGPPGPPGSPGVNGQKGIPGDPAFGHLGPPGKRGLSGVPGIKGPRGDPGCPGAEGPAGIPGFLGLKGPKGREGHAGFPGVPGPPGHSCERGAPGIPGQPGLPGYPGSPGAPGGKGQPGDVGPPGPAGMKGLPGLPGRPGAHGPPGLPGIPGPFGDDGLPGPPGPKGPRGLPGFPGFPGERGKPGAEGCPGAKGEPGEKGMSGLPGDRGLRGAKGAIGPPGDEGEMAIISQKGTPGEPGPPGDDGFPGERGDKGTPGMQGRRGEPGRYGPPGFHRGEPGEKGQPGPPGPPGPPGSTGLRGFIGFPGLPGDQGEPGSPGPPGFSGIDGARGPKGNKGDPASHFGPPGPKGEPGSPGCPGHFGASGEQGLPGIQGPRGSPGRPGPPGSSGPPGCPGDHGMPGLRGQPGEMGDPGPRGLQGDPGIPGPPGIKGPSGSPGLNGLHGLKGQKGTKGASGLHDVGPPGPVGIPGLKGERGDPGSPGISPPGPRGKKGPPGPPGSSGPPGPAGATGRAPKDIPDPGPPGDQGPPGPDGPRGAPGPPGLPGSVDLLRGEPGDCGLPGPPGPPGPPGPPGYKGFPGCDGKDGQKGPVGFPGPQGPHGFPGPPGEKGLPGPPGRKGPTGLPGPRGEPGPPADVDDCPRIPGLPGAPGMRGPEGAMGLPGMRGPSGPGCKGEPGLDGRRGVDGVPGSPGPPGRKGDTGEDGYPGGPGPPGPIGDPGPKGFGPGYLGGFLLVLHSQTDQEPTCPLGMPRLWTGYSLLYLEGQEKAHNQDLGLAGSCLPVFSTLPFAYCNIHQVCHYAQRNDRSYWLASAAPLPMMPLSEEAIRPYVSRCAVCEAPAQAVAVHSQDQSIPPCPQTWRSLWIGYSFLMHTGAGDQGGGQALMSPGSCLEDFRAAPFLECQGRQGTCHFFANKYSFWLTTVKADLQFSSAPAPDTLKESQAQRQKI SRCQVCVKYS 96human alpha5 MEVDSGKTENRDWEGFCYSTSAYWKNLYDGLLACYGCSPGSKCDCSGIKGEKGERGchain of type FPGLEGHPGLPGFPGPEGPPGPRGQKGDDGIPGPPGPKGIRGPPGLPGFPGTPGLPIV collagen GMPGHDGAPGPQGIPGCNGTKGERGFPGSPGFPGLQGPPGPPGIPGMKGEPGSIIM(NCBI SSLPGPKGNPGYPGPPGIQGLPGPTGIPGPIGPPGPPGLMGPPGPPGLPGPKGNMG ReferenceLNFQGPKGEKGEQGLQGPPGPPGQISEQKRPIDVEFQKGDQGLPGDRGPPGPPGIR Sequence:GPPGPPGGEKGEKGEQGEPGKRGKPGKDGENGQPGIPGLPGDPGYPGEPGRDGEKG XP_011529151.2)QKGDTGPPGPPGLVIPRPGTGITIGEKGNIGLPGLPGEKGERGFPGIQGPPGLPGPPGAAVMGPPGPPGFPGERGQKGDEGPPGISIPGPPGLDGQPGAPGLPGPPGPAGPHIPPSDEICEPGPPGPPGSPGDKGLQGEQGVKGDKGDTCFNCIGTGISGPPGQPGLPGLPGPPGSLGFPGQKGEKGQAGATGPKGLPGIPGAPGAPGFPGSKGEPGDILTFPGMKGDKGELGSPGAPGLPGLPGTPGQDGLPGLPGPKGEPGGITFKGERGPPGNPGLPGLPGNIGPMGPPGFGPPGPVGEKGIQGVAGNPGQPGIPGPKGDPGQTITQPGKPGLPGNPGRDGDVGLPGDPGLPGQPGLPGIPGSKGEPGIPGIGLPGPPGPKGFPGIPGPPGAPGTPGRIGLEGPPGPPGFPGPKGEPGFALPGPPGPPGLPGFKGALGPKGDRGFPGPPGPPGRTGLDGLPGPKGDVGPNGQPGPMGPPGLPGIGVQGPPGPPGIPGPIGQPGLHGIPGEKGDPGPPGLDVPGPPGERGSPGIPGAPGPIGPPGSPGLPGKAGASGFPGTKGEMGMMGPPGPPGPLGIPGRSGVPGLKGDDGLQGQPGLPGPTGEKGSKGEPGLPGPPGPMDPNLLGSKGEKGEPGLPGIPGVSGPKGYQGLPGDPGQPGLSGQPGLPGPPGPKGNPGLPGQPGLIGPPGLKGTIGDMGFPGPQGVEGPPGPSGVPGQPGSPGLPGQKGDKGDPGISSIGLPGIPGPKGEPGLPGYPGNPGIKGSVGDPGLPGLPGTPGAKGQPGLPGFPGTPGPPGPKGISGPPGNPGLPGEPGPVGGGGHPGQPGPPGEKGKPGQDGIPGPAGQKGEPGQPGFGNPGPPGLPGLSGQKGDGGLPGIPGNPGLPGPKGEPGFHGFPGVQGPPGPPGSPGPALEGPKGNPGPQGPPGRPGPTGFQGLPGPEGPPGLPGNGGIKGEKGNPGQPGLPGLPGLKGDQGPPGLQGNPGRPGLNGMKGDPGLPGVPGFPGMKGPSGVPGSAGPEGEPGLIGPPGPPGLPGPSGQSIIIKGDAGPPGIPGQPGLKGLPGPQGPQGLPGPTGPPGDPGRNGLPGFDGAGGRKGDPGLPGQPGTRGLDGPPGPDGLQGPPGPPGTSSVAHGFLITRHSQTTDAPQCPQGTLQVYEGFSLLYVQGNKRAHGQDLGTAGSCLRRFSTMPFMFCNINNVCNFASRNDYSYWLSTPEPMPMSMQPLKGQSIQPFISRCAVCEAPAVVIAVHSQTIQIPHCPQGWDSLWIGYSFMMHTSAGAEGSGQALASPGSCLEEFRSAPFIECHGRGTCNYYANSYSFWLATVDVSDMFSKPQSETLKAGDLRTRISRCQVCMKRT 97 human alpha6MLINKLWLLLVTLCLTEELAAAGEKSYGKPCGGQDCSGSCQCFPEKGARGRPGPIG chain of typeIQGPTGPQGFTGSTGLSGLKGERGFPGLLGPYGPKGDKGPMGVPGFLGINGIPGHP IV collagenGQPGPRGPPGLDGCNGTQGAVGFPGPDGYPGLLGPPGLPGQKGSKGDPVLAPGSFK (NCBIGMKGDPGLPGLDGITGPQGAPGFPGAVGPAGPPGLQGPPGPPGPLGPDGNMGLGFQ ReferenceGEKGVKGDVGLPGPAGPPPSTGELEFMGFPKGKKGSKGEPGPKGFPGISGPPGFPG Sequence:LGTTGEKGEKGEKGIPGLPGPRGPMGSEGVQGPPGQQGKKGTLGFPGLNGFQGIEG XP_006724680.1)QKGDIGLPGPDVFIDIDGAVISGNPGDPGVPGLPGLKGDEGIQGLRGPSGVPGLPALSGVPGALGPQGFPGLKGDQGNPGRTTIGAAGLPGRDGLPGPPGPPGPPSPEFETETLHNKESGFPGLRGEQGPKGNLGLKGIKGDSGFCACDGGVPNTGPPGEPGPPGPWGLIGLPGLKGARGDRGSGGAQGPAGAPGLVGPLGPSGPKGKKGEPILSTIQGMPGDRGDSGSQGFRGVIGEPGKDGVPGLPGLPGLPGDGGQGFPGEKGLPGLPGEKGHPGPPGLPGNGLPGLPGPRGLPGDKGKDGLPGQQGLPGSKGDCCCREVGKGDLDTERGITLPCIIPGSYGPSGFPGTPGFPGPKGSRGLPGTPGQPGSSGSKGEPGSPGLVHLPELPGFPGPRGEKGLPGFPGLPGKDGLPGMIGSPGLPGSKGATGDIFGAENGAPGEQGLQGLTGHKGFLGDSGLPGLKGVHGKPGLLGPKGERGSPGTPGQVGQPGTPGSSGPYGIKGKSGLPGAPGFPGISGHPGKKGTRGKKGPPGSIVKKGLPGLKGLPGNPGLVGLKGSPGSPGVAGLPALSGPKGEKGSVGFVGFPGIPGLPGIPGTRGLKGIPGSTGKMGPSGRAGTPGEKGDRGNPGPVGIPSPRRPMSNLWLKGDKGSQGSAGSNGFPGPRGDKGEAGRPGPPGLPGAPGLPGIIKGVSGKPGPPGFMGIRGLPGLKGSSGITGFPGMPGESGSQGIRGSPGLPGASGLPGLKGDNGQTVEISGSPGPKGQPGESGFKGTKGRDGLIGNIGFPGNKGEDGKVGVSGDVGLPGAPGFPGVAGMRGEPGLPGSSGHQGAIGPLGSPGLIGPKGFPGFPGLHGLNGLPGTKGTHGTPGPSITGVPGPAGLPGPKGEKGYPGIGIGAPGKPGLRGQKGDRGFPGLQGPAGLPGAPGISLPSLIAGQPGDPGRPGLDGERGRPGPAGPPGPPGPSSNQGDTGDPGFPGIPGPKGPKGDQGIPGFSGLPGELGLKGMRGEPGFMGTPGKVGPPGDPGFPGMKGKAGPRGSSGLQGDPGQTPTAEAVQVPPGPLGLPGIDGIPGLTGDPGAQGPVGLQGSKGLPGIPGKDGPSGLPGPPGALGDPGLPGLQGPPGFEGAPGQQGPFGMPGMPGQSMRVGYTLVKHSQSEQVPPCPIGMSQLWVGYSLLFVEGQEKAHNQDLGFAGSCLPRFSTMPFIYCNINEVCHYARRNDKSYWLSTTAPIPMMPVSQTQIPQYISRCSVCEAPSQAIAVHSQDITIPQCPLGWRSLWIGYSFLMHTAAGAEGGGQSLVSPGSCLEDFRATPFIECSGARGTCHYFANKYSFWLTTVEERQQFGELPVSETLKAGQLHTRVSRCQVCMKSL 98 LAIR-1QEEDLPRPSISAEPGTVIPLGSHVTFVCRGPVGVQTFRLERESRSTYNDTEDVSQASPSESEARFRIDSVSEGNAGPYRCIYYKPPKWSEQSDYLELLVKETSGGPDSPDTEPGSSAGPTQRPSDNSHNEHAPASQGLKAEHLYILIGVSVVFLFCLLLLVLFCLHRQNQIKQGPPRSKDEEQKPQQRPDLAVDVLERTADKATVNGLPEKDRETDTSALAAGSSQEVTYAQLDHWALTQRTARAVSPQSTKPMAESITYAAVARH 99 LAIR-2QEGALPRPSISAEPGTVISPGSHVTFMCRGPVGVQTFRLEREDRAKYKDSYNVFRLGPSESEARFHIDSVSEGNAGLYRCLYYKPPGWSEHSDFLELLVKESSGGPDSPDTEPGSSAGTVPGTEASGFDAP 100 GlycoproteinGDLLIQKTIKKQVVLEEGTIAFKNWVKTGTEVYRQFWIFDVQNPQEVMMNSSNIQV IV (CD36KQRGPYTYRVRFLAKENVTQDAEDNTVSFLQPNGAIFEPSLSVGTEADNFTVLNLA extracellularVAAASHIYQNQFVQMILNSLINKSKSSMFQVRTLRELLWGYRDPFLSLVPYPVTTT domain)VGLFYPYNNTADGVYKVFNGKDNISKVAIIDTYKGKRNLSYWESHCDMINGTDAASFPPFVEKSQVLQFFSSDICRSIYAVFESDVNLKGIPVYRFVLPSKAFASPVENPDNYCFCTEKIISKNCTSYGVLDISKCKEGRPVYISLPHFLYASPDVSEPIDGLNPNEEEHRTYLDIEPITGFTLQFAKRLQVNLLVKPSEKIQVLKNLKRNYIVPILWLNETGTIGDEKANMFRSQVTGKIN 101 NidogenLSRQELFPFGPGQGDLELEDGDDFVSPALELSGALRFYDRSDIDAVYVTTNGIIATSEPPAKESHPGLFPPTFGAVAPFLADLDTTDGLGKVYYREDLSPSITQRAAECVHRGFPEISFQPSSAVVVTWESVAPYQGPSRDPDQKGKRNTFQAVLASSDSSSYAIFLYPEDGLQFHTTFSKKENNQVPAVVAFSQGSVGFLWKSNGAYNIFANDRESVENLAKSSNSGQQGVWVFEIGSPATTNGVVPADVILGTEDGAEYDDEDEDYDLATTRLGLEDVGTTPFSYKALRRGGADTYSVPSVLSPRRAATERPLGPPTERTRSFQLAVETFHQQHPQVIDVDEVEETGVVFSYNTDSRQTCANNRHQCSVHAECRDYATGFCCSCVAGYTGNGRQCVAEGSPQRVNGKVKGRIFVGSSQVPIVFENTDLHSYVVMNHGRSYTAISTIPETVGYSLLPLAPVGGIIGWMFAVEQDGFKNGFSITGGEFTRQAEVTFVGHPGNLVIKQRFSGIDEHGHLTIDTELEGRVPQIPFGSSVHIEPYTELYHYSTSVITSSSTREYTVTEPERDGASPSRIYTYQWRQTITFQECVHDDSRPALPSTQQLSVDSVFVLYNQEEKILRYALSNSIGPVREGSPDALQNPCYIGTHGCDTNAACRPGPRTQFTCECSIGFRGDGRTCYDIDECSEQPSVCGSHTICNNHPGTFRCECVEGYQFSDEGTCVAVVDQRPINYCETGLHNCDIPQRAQCIYTGGSSYTCSCLPGFSGDGQACQDVDECQPSRCHPDAFCYNTPGSFTCQCKPGYQGDGFRCVPGEVEKTRCQHEREHILGAAGATDPQRPIPPGLFVPECDAHGHYAPTQCHGSTGYCWCVDRDGREVEGTRTRPGMTPPCLSTVAPPIHQGPAVPTAVIPLPPGTHLLFAQTGKIERLPLEGNTMRKTEAKAFLHVPAKVIIGLAFDCVDKMVYWTDITEPSIGRASLHGGEPTTIIRQDLGSPEGIAVDHLGRNIFWTDSNLDRIEVAKLDGTQRRVLFETDLVNPRGIVTDSVRGNLYWTDWNRDNPKIETSYMDGTNRRILVQDDLGLPNGLTFDAFSSQLCWVDAGTNRAECLNPSQPSRRKALEGLQYPFAVTSYGKNLYFTDWKMNSVVALDLAISKETDAFQPHKQTRLYGITTALSQCPQGHNYCSVNNGGCTHLCLATPGSRTCRCPDNTLGVDCIEQK 102 PerlecanVTHGLRAYDGLSLPEDIETVTASQMRWTHSYLSDDEDMLADSISGDDLGSGDLGSGDFQMVYFRALVNFTRSIEYSPQLEDAGSREFREVSEAVVDTLESEYLKIPGDQVVSVVFIKELDGWVFVELDVGSEGNADGAQIQEMLLRVISSGSVASYVTSPQGFQFRRLGTVPQFPRACTEAEFACHSYNECVALEYRCDRRPDCRDMSDELNCEEPVLGISPTFSLLVETTSLPPRPETTIMRQPPVTHAPQPLLPGSVRPLPCGPQEAACRNGHCIPRDYLCDGQEDCEDGSDELDCGPPPPCEPNEFPCGNGHCALKLWRCDGDFDCEDRTDEANCPTKRPEEVCGPTQFRCVSTNMCIPASFHCDEESDCPDRSDEFGCMPPQVVTPPRESIQASRGQTVTFTCVAIGVPTPIINWRLNWGHIPSHPRVTVTSEGGRGTLIIRDVKESDQGAYTCEAMNARGMVFGIPDGVLELVPQRGPCPDGHFYLEHSAACLPCFCFGITSVCQSTRRFRDQIRLRFDQPDDFKGVNVTMPAQPGTPPLSSTQLQIDPSLHEFQLVDLSRRFLVHDSFWALPEQFLGNKVDSYGGSLRYNVRYELARGMLEPVQRPDVVLMGAGYRLLSRGHTPTQPGALNQRQVQFSEEHWVHESGRPVQRAELLQVLQSLEAVLIQTVYNTKMASVGLSDIAMDTTVTHATSHGRAHSVEECRCPIGYSGLSCESCDAHFTRVPGGPYLGTCSGCNCNGHASSCDPVYGHCLNCQHNTEGPQCNKCKAGFFGDAMKATATSCRPCPCPYIDASRRFSDTCFLDTDGQATCDACAPGYTGRRCESCAPGYEGNPIQPGGKCRPVNQEIVRCDERGSMGTSGEACRCKNNVVGRLCNECADGSFHLSTRNPDGCLKCFCMGVSRHCTSSSWSRAQLHGASEEPGHFSLTNAASTHTTNEGIFSPTPGELGFSSFHRLLSGPYFWSLPSRFLGDKVTSYGGELRFTVTQRSQPGSTPLHGQPLVVLQGNNIILEHHVAQEPSPGQPSTFIVPFREQAWQRPDGQPATREHLLMALAGIDTLLIRASYAQQPAESRVSGISMDVAVPEETGQDPALEVEQCSCPPGYRGPSCQDCDTGYTRTPSGLYLGTCERCSCHGHSEACEPETGACQGCQHHTEGPRCEQCQPGYYGDAQRGTPQDCQLCPCYGDPAAGQAAHTCFLDTDGHPTCDACSPGHSGRHCERCAPGYYGNPSQGQPCQRDSQVPGPIGCNCDPQGSVSSQCDAAGQCQCKAQVEGLTCSHCRPHHFHLSASNPDGCLPCFCMGITQQCASSAYTRHLISTHFAPGDFQGFALVNPQRNSRLTGEFTVEPVPEGAQLSFGNFAQLGHESFYWQLPETYQGDKVAAYGGKLRYTLSYTAGPQGSPLSDPDVQITGNNIMLVASQPALQGPERRSYEIMFREEFWRRPDGQPATREHLLMALADLDELLIRATFSSVPLAASISAVSLEVAQPGPSNRPRALEVEECRCPPGYIGLSCQDCAPGYTRTGSGLYLGHCELCECNGHSDLCHPETGACSQCQHNAAGEFCELCAPGYYGDATAGTPEDCQPCACPLTNPENMFSRTCESLGAGGYRCTACEPGYTGQYCEQCGPGYVGNPSVQGGQCLPETNQAPLVVEVHPARSIVPQGGSHSLRCQVSGSPPHYFYWSREDGRPVPSGTQQRHQGSELHFPSVQPSDAGVYICTCRNLHQSNTSRAELLVTEAPSKPITVTVEEQRSQSVRPGADVTFICTAKSKSPAYTLVWTRLHNGKLPTRAMDFNGILTIRNVQLSDAGTYVCTGSNMFAMDQGTATLHVQASGTLSAPVVSIHPPQLTVQPGQLAEFRCSATGSPTPTLEWTGGPGGQLPAKAQIHGGILRLPAVEPTDQAQYLCRAHSSAGQQVARAVLHVHGGGGPRVQVSPERTQVHAGRTVRLYCRAAGVPSATITWRKEGGSLPPQARSERTDIATLLIPAITTADAGFYLCVATSPAGTAQARIQVVVLSASDASPPPVKIESSSPSVTEGQTLDLNCVVAGSAHAQVTWYRRGGSLPPHTQVHGSRLRLPQVSPADSGEYVCRVENGSGPKEASITVSVLHGTHSGPSYTPVPGSTRPIRIEPSSSHVAEGQTLDLNCVVPGQAHAQVTWHKRGGSLPARHQTHGSLLRLHQVTPADSGEYVCHVVGTSGPLEASVLVTIEASVIPGPIPPVRIESSSSTVAEGQTLDLSCVVAGQAHAQVTWYKRGGSLPARHQVRGSRLYIFQASPADAGQYVCRASNGMEASITVTVTGTQGANLAYPAGSTQPIRIEPSSSQVAEGQTLDLNCVVPGQSHAQVTWHKRGGSLPVRHQTHGSLLRLYQASPADSGEYVCRVLGSSVPLEASVLVTIEPAGSVPALGVTPTVRIESSSSQVAEGQTLDLNCLVAGQAHAQVTWHKRGGSLPARHQVHGSRLRLLQVTPADSGEYVCRVVGSSGTQEASVLVTIQQRLSGSHSQGVAYPVRIESSSASLANGHTLDLNCLVASQAPHTITWYKRGGSLPSRHQIVGSRLRIPQVTPADSGEYVCHVSNGAGSRETSLIVTIQGSGSSHVPSVSPPIRIESSSPTVVEGQTLDLNCVVARQPQAIITWYKRGGSLPSRHQTHGSHLRLHQMSVADSGEYVCRANNNIDALEASIVISVSPSAGSPSAPGSSMPIRIESSSSHVAEGETLDLNCVVPGQAHAQVTWHKRGGSLPSHHQTRGSRLRLHHVSPADSGEYVCRVMGSSGPLEASVLVTIEASGSSAVHVPAPGGAPPIRIEPSSSRVAEGQTLDLKCVVPGQAHAQVTWHKRGGNLPARHQVHGPLLRLNQVSPADSGEYSCQVTGSSGTLEASVLVTIEPSSPGPIPAPGLAQPIYIEASSSHVTEGQTLDLNCVVPGQAHAQVTWYKRGGSLPARHQTHGSQLRLHLVSPADSGEYVCRAASGPGPEQEASFTVTVPPSEGSSYRLRSPVISIDPPSSTVQQGQDASFKCLIHDGAAPISLEWKTRNQELEDNVHISPNGSIITIVGTRPSNHGTYRCVASNAYGVAQSVVNLSVHGPPTVSVLPEGPVWVKVGKAVTLECVSAGEPRSSARWTRISSTPAKLEQRTYGLMDSHAVLQISSAKPSDAGTYVCLAQNALGTAQKQVEVIVDTGAMAPGAPQVQAEEAELTVEAGHTATLRCSATGSPAPTIHWSKLRSPLPWQHRLEGDTLIIPRVAQQDSGQYICNATSPAGHAEATIILHVESPPYATTVPEHASVQAGETVQLQCLAHGTPPLTFQWSRVGSSLPGRATARNELLHFERAAPEDSGRYRCRVTNKVGSAEAFAQLLVQGPPGSLPATSIPAGSTPTVQVTPQLETKSIGASVEFHCAVPSDRGTQLRWFKEGGQLPPGHSVQDGVLRIQNLDQSCQGTYICQAHGPWGKAQASAQLVIQALPSVLINIRTSVQTVVVGHAVEFECLALGDPKPQVTWSKVGGHLRPGIVQSGGVVRIAHVELADAGQYRCTATNAAGTTQSHVLLLVQALPQISMPQEVRVPAGSAAVFPCIASGYPTPDISWSKLDGSLPPDSRLENNMLMLPSVRPQDAGTYVCTATNRQGKVKAFAHLQVPERVVPYFTQTPYSFLPLPTIKDAYRKFEIKITFRPDSADGMLLYNGQKRVPGSPTNLANRQPDFISFGLVGGRPEFRFDAGSGMATIRHPTPLALGHFHTVTLLRSLTQGSLIVGDLAPVNGTSQGKFQGLDLNEELYLGGYPDYGAIPKAGLSSGFIGCVRELRIQGEEIVFHDLNLTAHGISHCPTCRDRPCQNGGQCHDSESSSYVCVCPAGFTGSRCEHSQALHCHPEACGPDATCVNRPDGRGYTCRCHLGRSGLRCEEGVTVTTPSLSGAGSYLALPALTNTHHELRLDVEFKPLAPDGVLLFSGGKSGPVEDFVSLAMVGGHLEFRYELGSGLAVLRSAEPLALGRWHRVSAERLNKDGSLRVNGGRPVLRSSPGKSQGLNLHTLLYLGGVEPSVPLSPATNMSAHFRGCVGEVSVNGKRLDLTYSFLGSQGIGQCYDSSPCERQPCQHGATCMPAGEYEFQCLCRDGFKGDLCEHEENPCQLREPCLHGGTCQGTRCLCLPGFSGPRCQQGSGHGIAESDWHLEGSGGNDAPGQYGAYFHDDGFLAFPGHVFSRSLPEVPETIELEVRTSTASGLLLWQGVEVGEAGQGKDFISLGLQDGHLVFRYQLGSGEARLVSEDPINDGEWHRVTALREGRRGSIQVDGEELVSGRSPGPNVAVNAKGSVYIGGAPDVATLTGGRFSSGITGCVKNLVLHSARPGAPPPQPLDLQHRAQAGANTRPC PS 103 BiglycanDEEASGADTSGVLDPDSVTPTYSAMCPFGCHCHLRVVQCSDLGLKSVPKEISPDTTLLDLQNNDISELRKDDFKGLQHLYALVLVNNKISKIHEKAFSPLRKLQKLYISKNHLVEIPPNLPSSLVELRIHDNRIRKVPKGVFSGLRNMNCIEMGGNPLENSGFEPGAFDGLKLNYLRISEAKLTGIPKDLPETLNELHLDHNKIQAIELEDLLRYSKLYRLGLGHNQIRMIENGSLSFLPTLRELHLDNNKLARVPSGLPDLKLLQVVYLHSNNITKVGVNDFCPMGFGVKRAYYNGISLFNNPVPYWEVQPATFRCVTDRLAIQFGNYKK 104 DecorinDEASGIGPEVPDDRDFEPSLGPVCPFRCQCHLRVVQCSDLGLDKVPKDLPPDTTLLDLQNNKITEIKDGDFKNLKNLHALILVNNKISKVSPGAFTPLVKLERLYLSKNQLKELPEKMPKTLQELRAHENEITKVRKVTFNGLNQMIVIELGTNPLKSSGIENGAFQGMKKLSYIRIADTNITSIPQGLPPSLTELHLDGNKISRVDAASLKGLNNLAKLGLSFNSISAVDNGSLANTPHLRELHLDNNKLTRVPGGLAEHKYIQVVYLHNNNISVVGSSDFCPPGHNTKKASYSGVSLFSNPVQYWEIQPSTFRCVYVRSAIQLGNYK 105 AsporinDMEDTDDDDDDDDDDDDDDEDNSLFPTREPRSHFFPFDLFPMCPFGCQCYSRVVHCSDLGLTSVPTNIPFDTRMLDLQNNKIKEIKENDFKGLTSLYGLILNNNKLTKIHPKAFLTTKKLRRLYLSHNQLSEIPLNLPKSLAELRIHENKVKKIQKDTFKGMNALHVLEMSANPLDNNGIEPGAFEGVTVFHIRIAEAKLTSVPKGLPPTLLELHLDYNKISTVELEDFKRYKELQRLGLGNNKITDIENGSLANIPRVREIHLENNKLKKIPSGLPELKYLQIIFLHSNSIARVGVNDFCPTVPKMKKSLYSAISLFNNPVKYWEMQPATFRCVL SRMSVQLGNFGM106 FibromodulinQYEDDPHWWFHYLRSQQSTYYDPYDPYPYETYEPYPYGVDEGPAYTYGSPSPPDPRDCPQECDCPPNFPTAMYCDNRNLKYLPFVPSRMKYVYFQNNQITSIQEGVFDNATGLLWIALHGNQITSDKVGRKVFSKLRHLERLYLDHNNLTRMPGPLPRSLRELHLDHNQISRVPNNALEGLENLTALYLQHNEIQEVGSSMRGLRSLILLDLSYNHLRKVPDGLPSALEQLYMEHNNVYTVPDSYFRGAPKLLYVRLSHNSLTNNGLASNTFNSSSLLELDLSYNQLQKIPPVNTNLENLYLQGNRINEFSISSFCTVVDVVNFSKLQVLRLDGNEIKRSAMPADAPLCLRLASLIEI 107 LumicanQYYDYDFPLSIYGQSSPNCAPECNCPESYPSAMYCDELKLKSVPMVPPGIKYLYLRNNQIDHIDEKAFENVTDLQWLILDHNLLENSKIKGRVFSKLKQLKKLHINHNNLTESVGPLPKSLEDLQLTHNKITKLGSFEGLVNLTFIHLQHNRLKEDAVSAAFKGLKSLEYLDLSFNQIARLPSGLPVSLLTLYLDNNKISNIPDEYFKRFNALQYLRLSHNELADSGIPGNSFNVSSLVELDLSYNKLKNIPTVNENLENYYLEVNQLEKFDIKSFCKILGPLSYSKIKHLRLDGNRISETSLPPDMYECLRVANEVTLN 108 PRELPQPTRRPRPGTGPGRRPRPRPRPTPSFPQPDEPAEPTDLPPPLPPGPPSIFPDCPRECYCPPDFPSALYCDSRNLRKVPVIPPRIHYLYLQNNFITELPVESFQNATGLRWINLDNNRIRKIDQRVLEKLPGLVFLYMEKNQLEEVPSALPRNLEQLRLSQNHISRIPPGVFSKLENLLLLDLQHNRLSDGVFKPDTFHGLKNLMQLNLAHNILRKMPPRVPTAIHQLYLDSNKIETIPNGYFKSFPNLAFIRLNYNKLTDRGLPKNSFNISNLLVLHLSHNRISSVPAINNRLEHLYLNNNSIEKINGTQICPNDLVAFHDFSSDLENVPHLRYLRLDGNYLKPPIPLDLMMCFRLLQSV

VI 109 Osteoadherin/QYETYQWDEDYDQEPDDDYQTGFPFRQNVDYGVPFHQYTLGCVSECFCPTNFPSSM OsteomodulinYCDNRKLKTIPNIPMHIQQLYLQFNEIEAVTANSFINATHLKEINLSHNKIKSQKIDYGVFAKLPNLLQLHLEHNNLEEFPFPLPKSLERLLLGYNEISKLQTNAMDGLVNLTMLDLCYNYLHDSLLKDKIFAKMEKLMQLNLCSNRLESMPPGLPSSLMYLSLENNSISSIPEKYFDKLPKLHTLRMSHNKLQDIPYNIFNLPNIVELSVGHNKLKQAFYIPRNLEHLYLQNNEIEKMNLTVMCPSIDPLHYHHLTYIRVDQNKLKEPISSYIFFCFPHIHTIYYGEQRSTNGQTIQLKTQVFRRFPDDDDESEDHDDPDNAHESPEQEGAEGHF DLHYYENQE 110Opticin ASLPRKERKRREEQMPREGDSFEVLPLRNDVLNPDNYGEVIDLSNYEELTDYGDQLPEVKVTSLAPATSISPAKSTTAPGTPSSNPTMTRPTTAGLLLSSQPNHGLPTCLVCVCLGSSVYCDDIDLEDIPPLPRRTAYLYARFNRISRIRAEDFKGLTKLKRIDLSNNLISSIDNDAFRLLHALQDLILPENQLEALPVLPSGIEFLDVRLNRLQSSGIQPAAFRAMEKLQFLYLSDNLLDSIPGPLPLSLRSVHLQNNLIETMQRDVFCDPEEHKHTRRQLEDIRLDGNPINLSLFPSAYFCLPRLPIGRFT 111 Osteoglycin/PPTQQDSRIIYDYGTDNFEESIFSQDYEDKYLDGKNIKEKETVIIPNEKSLQLQKD MimecanEAITPLPPKKENDEMPTCLLCVCLSGSVYCEEVDIDAVPPLPKESAYLYARFNKIKKLTAKDFADIPNLRRLDFTGNLIEDIEDGTFSKLSLLEELSLAENQLLKLPVLPPKLTLFNAKYNKIKSRGIKANAFKKLNNLTFLYLDHNALESVPLNLPESLRVIHLQFNNIASITDDTFCKANDTSYIRDRIEEIRLEGNPIVLGKHPNSFICLKRLPIGSYF 112Chondroadherin QRCPQACICDNSRRHVACRYQNLTEVPDAIPELTQRLDLQGNLLKVIPAAAFQGVPHLTHLDLRHCEVELVAEGAFRGLGRLLLLNLASNHLRELPQEALDGLGSLRRLELEGNALEELRPGTFGALGALATLNLAHNALVYLPAMAFQGLLRVRWLRLSHNALSVLAPEALAGLPALRRLSLHHNELQALPGPVLSQARGLARLELGHNPLTYAGEEDGLALPGLRELLLDGGALQALGPRAFAHCPRLHTLDLRGNQLDTLPPLQGPGQLRRLRLQGNPLWCGCQARPLLEWLARARVRSDGACQGPRRLRGEALDALRPWDLRCPGDAAQEEEELEERAVAGPRAPPRGPPRGPGEERAVAPCPRACVCVPESRHSSCEGCGLQAVPRGFPSDTQLLDLRRNHFPSVPRAAFPGLGHLVSLHLQHCGIAELEAGALAGLGRLIYLYLSDNQLAGLSAAALEGAPRLGYLYLERNRFLQVPGAALRALPSLFSLHLQDNAVDRLAPGDLGRTRALRWVYLSGNRITEVSLGALGPARELEKLHLDRNQLREVPTGALEGLPALLELQLSGNPLRALRDGAFQPVGRSLQHLFLNSSGLEQICPGAFSGLGPGLQSLHLQKNQLRALPALPSLSQLELIDLSSNPFHCDCQLLPLHRWLTGLNLRVGATCATPPNARGQRVKAAAAVFEDCPGWAARKAKRTPASRPSARRTPIKGRQCGADKVGKE KGRL 113Podocan GPVLAVRAPGFGRSGGHSLSPEENEFAEEEPVLVLSPEEPGPGPAAVSCPRDCACSQEGVVDCGGIDLREFPGDLPEHTNHLSLQNNQLEKIYPEELSRLHRLETLNLQNNRLTSRGLPEKAFEHLYNLNYLYLANNKLTLAPRFLPNALISVDFAANYLTKIYGLTFGQKPNLRSVYLHNNKLADAGLPDNMFNGSSNVEVLILSSNFLRHVPKHLPPALYKLHLKNNKLEKIPPGAFSELSSLRELYLQNNYLTDEGLDNETFWKLSSLEYLDLSSNNLSRVPAGLPRSLVLLHLEKNAIRSVDANVLTPIRSLEYLLLHSNQLREQGIHPLAFQGLKRLHTVHLYNNALERVPSGLPRRVRTLMILHNQITGIGREDFATTYFLEELNLSYNRITSPQVHRDAFRKLRLLRSLDLSGNRLHTLPPGLPRNVHVLKVKRNELAALARGALVGMAQLRELYLTSNRLRSRALGPRAWVDLAHLQLLDIAGNQLTEIPEGLPESLEYLYLQNNKISAVPANAFDSTPNLKGIFLRFNKLAVGSVVDSAFRRLKHLQVLDIEGNLEFGDISKDRGRLGKEKEEEEEEEEEEEETR 114 Human IgG1ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAV constantLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPC region (aminoPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVH acid sequence)NAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 115 Human IgG1EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDP Fc domainEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKA (amino acidLPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWES sequence)NGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQK SLSLSPGK 116HSA domain I DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQR 117 HSA domain IIGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQ 118 HSA domain IIINLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGL 119 LinkerLEA (EAAAK) ₄ALEA (EAAAK) ₄ALE 120 Lumican-QYYDYDIPLFMYGQISPNCAPECNCPHSYPTAMYCDDLKLKSVPMVPPGIKYLYLR MSA-IL2NNQIDHIDEKAFENVTDLQWLILDHNLLENSKIKGKVFSKLKQLKKLHINYNNLTE (lumican inSVGPLPKSLQDLQLTNNKISKLGSFDGLVNLTFIYLQHNQLKEDAVSASLKGLKSL bold; linkersEYLDLSFNQMSKLPAGLPTSLLTLYLDNNKISNIPDEYFKRFTGLQYLRLSHNELA in italics;DSGVPGNSFNISSLLELDLSYNKLKSIPTVNENLENYYLEVNELEKFDVKSFCKIL MSA underlined;GPLSYSKIKHLRLDGNPLTQSSLPPDMYECLRVANEITVN GGGSGGGS EAHKSEIA IL2 bold andHRYNDLGEQHFKGLVLIAFSQYLQKCSYDEHAKLVQEVTDFAKTCVADESAANCDK underline;SLHTLFGDKLCAIPNLRENYGELADCCTKQEPERNECFLQHKDDNPSLPPFERPEA HIS tag dottedEAMCTSFKENPTTFMGHYLHEVARRHPYFYAPELLYYAEQYNEILTQCCAEADKES underlined)CLTPKLDGVKEKALVSSVRQRMKCSSMQKFGERAFKAWAVARLSQTFPNADFAEITKLATDLTKVNKECCHGDLLECADDRAELAKYMCENQATISSKLQTCCDKPLLKKAHCLSEVEHDTMPADLPAIAADFVEDQEVCKNYAEAKDVFLGTFLYEYSRRHPDYSVSLLLRLAKKYEATLEKCCAEANPPACYGTVLAEFQPLVEEPKNLVKTNCDLYEKLGEYGFQNAILVRYTQKAPQVSTPTLVEAARNLGRVGTKCCTLPEDQRLPCVEDYLSAILNRVCLLHEKTPVSEHVTKCCSGSLVERRPCFSALTVDETYVPKEFKAETFTFHSDICTLPEKEKQIKKQTALAELVKHKPKATAEQLKTVMDDFAQFLDTCCKAADKDTCFSTEGPNLVTRCKDALA GGGS APTSSSTSSSTAEAQQQQQQQQQQQQHLEQLLMDLQELLSRMENYRNLKLPRMLTFKFYLPKQATELKDLQCLEDELGPLRHVLDLTQSKSFQLEDAENFISNIRVTVVKLKGSDNTFECQFDDESATVVDFLRRWIAFCQSIISTSP Q

121 MSA-IL2 EAHKSEIAHRYNDLGEQHFKGLVLIAFSQYLQKCSYDEHAKLVQEVTDFAKTCVAD(MSA in ESAANCDKSLHTLFGDKLCAIPNLRENYGELADCCTKQEPERNECFLQHKDDNPSLbold; linkers PPFERPEAEAMCTSFKENPTTFMGHYLHEVARRHPYFYAPELLYYAEQYNEILTQCin italics; CAEADKESCLTPKLDGVKEKALVSSVRQRMKCSSMQKFGERAFKAWAVARLSQTFPIL2 underlined; NADFAEITKLATDLTKVNKECCHGDLLECADDRAELAKYMCENQATISSKLQTCCDHIS tag dotted KPLLKKAHCLSEVEHDTMPADLPAIAADFVEDQEVCKNYAEAKDVFLGTFLYEYSRunderlined) RHPDYSVSLLLRLAKKYEATLEKCCAEANPPACYGTVLAEFQPLVEEPKNLVKTNCDLYEKLGEYGFQNAILVRYTQKAPQVSTPTLVEAARNLGRVGTKCCTLPEDQRLPCVEDYLSAILNRVCLLHEKTPVSEHVTKCCSGSLVERRPCFSALTVDETYVPKEFKAETFTFHSDICTLPEKEKQIKKQTALAELVKHKPKATAEQLKTVMDDFAQFLDTCCKAADKDTCFSTEGPNLVTRCKDALA GGGS APTSSSTSSSTAEAQQQQQQQQQQQQHLEQLLMDLQELLSRMENYRNLKLPRMLTFKFYLPKQATELKDLQCLEDELGPLRHVLDLTQSKSFQLEDAENFISNIRVTVVKLKGSDNTFECQFDDESATVVDFLRRWIAFC QSIISTSPQ

122 IL12-MSA MWELEKDVYVVEVDWTPDAPGETVNLTCDTPEEDDITWTSDQRHGVIGSGKTLTIT(IL12p40 bold; VKEFLDAGQYTCHKGGETLSHSHLLLHKKENGIWSTEILKNFKNKTFLKCEAPNYSlinkers  GRFTCSWLVQRNMDLKFNIKSSSSSPDSRAVTCGMASLSAEKVTLDQRDYEKYSVSitalics; CQEDVTCPTAEETLPIELALEARQQNKYENYSTSFFIRDIIKPDPPKNLQMKPLKNIL12p34 SQVEVSWEYPDSWSTPHSYFSLKFFVRIQRKKEKMKETEEGCNQKGAFLVEKTSTEunderlined; VQCKGGNVCVQAQDRYYNSSCSKWACVPCRVRS GGSGGGSGGGSGGGS RVIPVSGPMSA bold and ARCLSQSRNLLKTTDDMVKTAREKLKHYSCTAEDIDHEDITRDQTSTLKTCLPLELunderlined; HKNESCLATRETSSTTRGSCLPPQKTSLMMTLCLGSIYEDLKMYQTEFQAINAALQHIS tag NHNHQQIILDKGMLVAIDELMQSLNHNGETLRQKPPVGEADPYRVKMKLCILLHAF dottedSTRVVTINRVMGYLSSA GSGGGS EAHKSEIAHRYNDLGEQHFKGLVLIAFSQYLQK underlined)CSYDEHAKLVQEVTDFAKTCVADESAANCDKSLHTLFGDKLCAIPNLRENYGELADCCTKQEPERNECFLQHKDDNPSLPPFERPEAEAMCTSFKENPTTFMGHYLHEVARRHPYFYAPELLYYAEQYNEILTQCCAEADKESCLTPKLDGVKEKALVSSVRQRMKCSSMQKFGERAFKAWAVARLSQTFPNADFAEITKLATDLTKVNKECCHGDLLECADDRAELAKYMCENQATISSKLQTCCDKPLLKKAHCLSEVEHDTMPADLPAIAADFVEDQEVCKNYAEAKDVFLGTFLYEYSRRHPDYSVSLLLRLAKKYEATLEKCCAEANPPACYGTVLAEFQPLVEEPKNLVKTNCDLYEKLGEYGFQNAILVRYTQKAPQVSTPTLVEAARNLGRVGTKCCTLPEDQRLPCVEDYLSAILNRVCLLHEKTPVSEHVTKCCSGSLVERRPCFSALTVDETYVPKEFKAETFTFHSDICTLPEKEKQIKKQTALAELVKHKPKATAEQLKTVMDDFAQFLDTCCKAADKDTCFSTEGPNLVTRCKDALA

123 IL12-MSA- MWELEKDVYVVEVDWTPDAPGETVNLTCDTPEEDDITWTSDQRHGVIGSGKTLTITLumican VKEFLDAGQYTCHKGGETLSHSHLLLHKKENGIWSTEILKNFKNKTFLKCEAPNYS(IL12p40 bold; GRFTCSWLVQRNMDLKFNIKSSSSSPDSRAVTCGMASLSAEKVTLDQRDYEKYSVSlinkers italics;CQEDVTCPTAEETLPIELALEARQQNKYENYSTSFFIRDIIKPDPPKNLQMKPLKN IL12p34SQVEVSWEYPDSWSTPHSYFSLKFFVRIQRKKEKMKETEEGCNQKGAFLVEKTSTE underlined;VQCKGGNVCVQAQDRYYNSSCSKWACVPCRVRS GGSGGGSGGGSGGGS RVIPVSGP MSA bold andARCLSQSRNLLKTTDDMVKTAREKLKHYSCTAEDIDHEDITRDQTSTLKTCLPLEL underlined;HKNESCLATRETSSTTRGSCLPPQKTSLMMTLCLGSIYEDLKMYQTEFQAINAALQ lumican bold,NHNHQQIILDKGMLVAIDELMQSLNHNGETLRQKPPVGEADPYRVKMKLCILLHAF underlinedSTRVVTINRVMGYLSSA GSGGGS EAHKSEIAHRYNDLGEQHFKGLVLIAFSQYLQKand italc; HIS CSYDEHAKLVQEVTDFAKTCVADESAANCDKSLHTLFGDKLCAIPNLRENYGELADtag dotted CCTKQEPERNECFLQHKDDNPSLPPFERPEAEAMCTSFKENPTTFMGHYLHEVARRunderlined) HPYFYAPELLYYAEQYNEILTQCCAEADKESCLTPKLDGVKEKALVSSVRQRMKCSSMQKFGERAFKAWAVARLSQTFPNADFAEITKLATDLTKVNKECCHGDLLECADDRAELAKYMCENQATISSKLQTCCDKPLLKKAHCLSEVEHDTMPADLPAIAADFVEDQEVCKNYAEAKDVFLGTFLYEYSRRHPDYSVSLLLRIAKKYEATLEKCCAEANPPACYGTVLAEFQPLVEEPKNLVKTNCDLYEKLGEYGFQNAILVRYTQKAPQVSTPTLVEAARNLGRVGTKCCTLPEDQRLPCVEDYLSAILNRVCLLHEKTPVSEHVTKCCSGSLVERRPCFSALTVDETYVPKEFKAETFTFHSDICTLPEKEKQIKKQTALAELVKHKPKATAEQLKTVMDDFAQFLDTCCKAADKDTCFSTEGPNLVTRCKDALA GGGSGGGS QYYDYDIPLFMYGQISPNCAPECNCPHSYPTAMYCDDLKLKSVPMVPPGIKYLYLRNNQIDHIDEKAFENVTDLQWLILDHNLLENSKIKGKVFSKLKQLKKLHINYNNLTESVGPLPKSLQDLQLTNNKISKLGSFDGLVNLTFIYLQHNQLKEDAVSASLKGLKSLEYLDLSFNQMSKLPAGLPTSLLTLYLDNNKISNIPDEYFKRFTGLQYLRLSHNELADSGVPGNSFNISSLLELDLSYNKLKSIPTVNENLENYYLEVNELEKFDVKSFCKILGPLSYSKIKHLRLDGNPLTQSSLPPDMYECLRVANEITVN

124 Lumican- QYYDYDIPLFMYGQISPNCAPECNCPHSYPTAMYCDDLKLKSVPMVPPGIKYLYLRGGGS-(H)₆ NNQIDHIDEKAFENVTDLQWLILDHNLLENSKIKGKVFSKLKQLKKLHINYNNLTESVGPLPKSLQDLQLTNNKISKLGSFDGLVNLTFIYLQHNQLKEDAVSASLKGLKSLEYLDLSFNQMSKLPAGLPTSLLTLYLDNNKISNIPDEYFKRFTGLQYLRLSHNELADSGVPGNSFNISSLLELDLSYNKLKSIPTVNENLENYYLEVNELEKFDVKSFCKILGPLSYSKIKHLRLDGNPLTQSSLPPDMYECLRVANEITVN GGGSHHHHHH 125 LumicanQYYDYDIPLFMYGQISPNCAPECNCPHSYPTAMYCDDLKLKSVPMVPPGIKYLYLR D213ANNQIDHIDEKAFENVTDLQWLILDHNLLENSKIKGKVFSKLKQLKKLHINYNNLTESVGPLPKSLQDLQLTNNKISKLGSFDGLVNLTFIYLQHNQLKEDAVSASLKGLKSLEYLDLSFNQMSKLPAGLPTSLLTLYLANNKISNIPDEYFKRFTGLQYLRLSHNELADSGVPGNSFNISSLLELDLSYNKLKSIPTVNENLENYYLEVNELEKFDVKSFCKILGPLSYSKIKHLRLDGNPLTQSSLPPDMYECLRVANEITVN GGGSHHHHHH 126 Lumican-MSAQYYDYDIPLFMYGQISPNCAPECNCPHSYPTAMYCDDLKLKSVPMVPPGIKYLYLRNNQIDHIDEKAFENVTDLQWLILDHNLLENSKIKGKVFSKLKQLKKLHINYNNLTESVGPLPKSLQDLQLTNNKISKLGSFDGLVNLTFIYLQHNQLKEDAVSASLKGLKSLEYLDLSFNQMSKLPAGLPTSLLTLYLDNNKISNIPDEYFKRFTGLQYLRLSHNELADSGVPGNSFNISSLLELDLSYNKLKSIPTVNENLENYYLEVNELEKFDVKSFCKILGPLSYSKIKHLRLDGNPLTQSSLPPDMYECLRVANEITVN GGGSGGGS EAHKSEIAHRYNDLGEQHFKGLVLIAFSQYLQKCSYDEHAKLVQEVTDFAKTCVADESAANCDKSLHTLFGDKLCAIPNLRENYGELADCCTKQEPERNECFLQHKDDNPSLPPFERPEAEAMCTSFKENPTTFMGHYLHEVARRHPYFYAPELLYYAEQYNEILTQCCAEADKESCLTPKLDGVKEKALVSSVRQRMKCSSMQKFGERAFKAWAVARLSQTFPNADFAEITKLATDLTKVNKECCHGDLLECADDRAELAKYMCENQATISSKLQTCCDKPLLKKAHCLSEVEHDTMPADLPAIAADFVEDQEVCKNYAEAKDVFLGTFLYEYSRRHPDYSVSLLLRLAKKYEATLEKCCAEANPPACYGTVLAEFQPLVEEPKNLVKTNCDLYEKLGEYGFQNAILVRYTQKAPQVSTPTLVEAARNLGRVGTKCCTLPEDQRLPCVEDYLSAILNRVCLLHEKTPVSEHVTKCCSGSLVERRPCFSALTVDETYVPKEFKAETFTFHSDICTLPEKEKQIKKQTALAELVKHKPKATAEQLKTVMDDFAQFLDTCCKAADKDTCFSTEGPNLVTRCKDALAHHHHHH  127 GlucKPTENNEDFNIVAVASNFATTDLDADRGKLPGKKLPLEVLKEMEANARKAGCTRGCLICLSHIKCTPKMKKFIPGRCHTYEGDKESAQGGIGEAIVDIPEIPGFKDLEPMEQFIAQVDLCVDCTTGCLKGLANVQCSDLLKKWLPQRCATFASKIQGQVDKIKGAGGD GGGSHHHHHH 128Lumican-Gluc QYYDYDIPLFMYGQISPNCAPECNCPHSYPTAMYCDDLKLKSVPMVPPGIKYLYLRNNQIDHIDEKAFENVTDLQWLILDHNLLENSKIKGKVFSKLKQLKKLHINYNNLTESVGPLPKSLQDLQLTNNKISKLGSFDGLVNLTFIYLQHNQLKEDAVSASLKGLKSLEYLDLSFNQMSKLPAGLPTSLLTLYLDNNKISNIPDEYFKRFTGLQYLRLSHNELADSGVPGNSFNISSLLELDLSYNKLKSIPTVNENLENYYLEVNELEKFDVKSFCKILGPLSYSKIKHLRLDGNPLTQSSLPPDMYECLRVANEITVN GGGSGGGGGGGS KPTENNEDFNIVAVASNFATTDLDADRGKLPGKKLPLEVLKEMEANARKAGCTRGCLICLSHIKCTPKMKKFIPGRCHTYEGDKESAQGGIGEAIVDIPEIPGFKDLEPMEQFIAQVDLCVDCTTGCLKGLANVQCSDLLKKWLPQRCATFASKIQGQVDKIKGAGGD GGGS HHHHHH 129CNA35-Gluc ARDISSTNVTDLTVSPSKIEDGGKTTVKMTFDDKNGKIQNGDMIKVAWPTSGTVKIEGYSKTVPLTVKGEQVGQAVITPDGATITFNDKVEKLSDVSGFAEFEVQGRNLTQTNTSDDKVATITSGNKSTNVTVHKSEAGTSSVFYYKTGDMLPEDTTHVRWFLNINNEKSYVSKDITIKDQIQGGQQLDLSTLNINVTGTHSNYYSGQSAITDFEKAFPGSKITVDNTKNTIDVTIPQGYGSYNSFSINYKTKITNEQQKEFVNNSQAWYQEHGKEEVNGKSFNHTVHNINANAGIEGTVKGELKVLKQDKDTK GGGSGGGGGGGG KPTENNEDFNIVAVASNFATTDLDADRGKLPGKKLPLEVLKEMEANARKAGCTRGCLICLSHIKCTPKMKKFIPGRCHTYEGDKESAQGGIGEAIVDIPEIPGFKDLEPMEQFIAQVDLCVDCTTGCLKGLANVQCSDLLKKWLPQRCATFASKIQGQVDKIKGAGGD GGGSHHHHHH 130ColG s3s/s3b- PITKEMEPNDDIKEANGPIVEGVTVKGDLNGSDDADTFYFDVKEDGDVTIELPYSGGluc SSNFTWLVYKEGDDQNHIASGIDKNNSKVGTFKSTKGRHYVFIYKHDSASNISYSLNIKGLGNEKLKEKENNDSSDKATVIPNFNTTMQGSLLGDDSRDYYSFEVKEEGEVNIELDKKDEFGVTWTLHPESNINDRITYGQVDGNKVSNKVKLRPGKYYLLVYKYSGS GNYELRVNGGGSGGGSGGGS KPTENNEDFNIVAVASNFATTDLDADRGKLPGKKLPLEVLKEMEANARKAGCTRGCLICLSHIKCTPKMKKFIPGRCHTYEGDKESAQGGIGEAIVDIPEIPGFKDLEPMEQFIAQVDLCVDCTTGCLKGLANVQCSDLLKKWLPQRCATFASKIQGQVDKIKGAGGD GGGSHHHHHH 131 ColH_s3-GlucGTEKEPNNSKETASGPIVPGIPVSGTIENTSDQDYFYFDVITPGEVKIDINKLGYGGATWVVYDENNNAVSYATDDGQNLSGKFKADKPGRYYIHLYMFNGSYMPYRINIE G GGSGGGSGGGSKPTENNEDFNIVAVASNFATTDLDADRGKLPGKKLPLEVLKEMEANARKAGCTRGCLICLSHIKCTPKMKKFIPGRCHTYEGDKESAQGGIGEAIVDIPEIPGFKDLEPMEQFIAQVDLCVDCTTGCLKGLANVQCSDLLKKWLPQRCATFASKIQG QVDKIKGAGGDGGGSHHHHHH 132 PLGF2 HBD- RRRPKGRGKRRREKQRPTDSHL GGGSGGGSGGGSKPTENNEDFNIVAVASNFATTD GlucLDADRGKLPGKKLPLEVLKEMEANARKAGCTRGCLICLSHIKCTPKMKKFIPGRCHTYEGDKESAQGGIGEAIVDIPEIPGFKDLEPMEQFIAQVDLCVDCTTGCLKGLANVQCSDLLKKWLPQRCATFASKIQGQVDKIKGAGGD GGGSHHHHHH 133 4M5.3-MSA-ADVVMTQTPLSLPVSLGDQASISCRSSQSLVHSNGNTYLRWYLQKPGQSPKVLIYK LumicanVSNRVSGVPDRFSGSGSGTDFTLKINRVEAEDLGVYFCSQSTHVPWTFGGGTKLEIKSSADDAKKDAAKKDDAKKDDAKKDGGVKLDETGGGLVQPGGAMKLSCVTSGFTFGHYWMNWVRQSPEKGLEWVAQFRNKPYNYETYYSDSVKGRFTISRDDSKSSVYLQMNNLRVEDTGIYYCTGASYGMEYLGQGTSVTVS GGGS EAHKSEIAHRYNDLGEQHFKGLVLIAFSQYLQKCSYDEHAKLVQEVTDFAKTCVADESAANCDKSLHTLFGDKLCAIPNLRENYGELADCCTKQEPERNECFLQHKDDNPSLPPFERPEAEAMCTSFKENPTTFMGHYLHEVARRHPYFYAPELLYYAEQYNEILTQCCAEADKESCLTPKLDGVKEKALVSSVRQRMKCSSMQKFGERAFKAWAVARLSQTFPNADFAEITKLATDLTKVNKECCHGDLLECADDRAELAKYMCENQATISSKLQTCCDKPLLKKAHCLSEVEHDTMPADLPAIAADFVEDQEVCKNYAEAKDVFLGTFLYEYSRRHPDYSVSLLLRLAKKYEATLEKCCAEANPPACYGTVLAEFQPLVEEPKNLVKTNCDLYEKLGEYGFQNAILVRYTQKAPQVSTPTLVEAARNLGRVGTKCCTLPEDQRLPCVEDYLSAILNRVCLLHEKTPVSEHVTKCCSGSLVERRPCFSALTVDETYVPKEFKAETFTFHSDICTLPEKEKQIKKQTALAELVKHKPKATAEQLKTVMDDFAQFLDTCCKAADKDTCFSTEGPNLVTRCKD ALA GGGSGGGSQYYDYDIPLFMYGQISPNCAPECNCPHSYPTAMYCDDLKLKSVPMVPPGIKYLYLRNNQIDHIDEKAFENVTDLQWLILDHNLLENSKIKGKVFSKLKQLKKLHINYNNLTESVGPLPKSLQDLQLTNNKISKLGSFDGLVNLTFIYLQHNQLKEDAVSASLKGLKSLEYLDLSFNQMSKLPAGLPTSLLTLYLDNNKISNIPDEYFKRFTGLQYLRLSHNELADSGVPGNSFNISSLLELDLSYNKLKSIPTVNENLENYYLEVNELEKFDVKSFCKILGPLSYSKIKHLRLDGNPLTQSSLPPDMYECLRVANEITVN GGGSH HHHHH 134Ss07d-MSA- ATVKFKYKGEEKQVDISKIYLVLRLGKFIYFYYDLGGGKLGLGHVSEKDAPKELLQLumican MLEKQKK GGGS EAHKSEIAHRYNDLGEQHFKGLVLIAFSQYLQKCSYDEHAKLVQEVTDFAKTCVADESAANCDKSLHTLFGDKLCAIPNLRENYGELADCCTKQEPERNECFLQHKDDNPSLPPFERPEAEAMCTSFKENPTTFMGHYLHEVARRHPYFYAPELLYYAEQYNEILTQCCAEADKESCLTPKLDGVKEKALVSSVRQRMKCSSMQKFGERAFKAWAVARLSQTFPNADFAEITKLATDLTKVNKECCHGDLLECADDRAELAKYMCENQATISSKLQTCCDKPLLKKAHCLSEVEHDTMPADLPAIAADFVEDQEVCKNYAEAKDVFLGTFLYEYSRRHPDYSVSLLLRLAKKYEATLEKCCAEANPPACYGTVLAEFQPLVEEPKNLVKTNCDLYEKLGEYGFQNAILVRYTQKAPQVSTPTLVEAARNLGRVGTKCCTLPEDQRLPCVEDYLSAILNRVCLLHEKTPVSEHVTKCCSGSLVERRPCFSALTVDETYVPKEFKAETFTFHSDICTLPEKEKQIKKQTALAELVKHKPKATAEQLKTVMDDFAQFLDTCCKAADKDTCFSTEGPNLVTRCKDALA GGGSGGGS QYYDYDIPLFMYGQISPNCAPECNCPHSYPTAMYCDDLKLKSVPMVPPGIKYLYLRNNQIDHIDEKAFENVTDLQWLILDHNLLENSKIKGKVFSKLKQLKKLHINYNNLTESVGPLPKSLQDLQLTNNKISKLGSFDGLVNLTFIYLQHNQLKEDAVSASLKGLKSLEYLDLSFNQMSKLPAGLPTSLLTLYLDNNKISNIPDEYFKRFTGLQYLRLSHNELADSGVPGNSFNISSLLELDLSYNKLKSIPTVNENLENYYLEVNELEKFDVKSFCKILGPLSYSKIKHLRLDGNPLTQSSLPPDMYECLRVANEITVN GGGSHHHHHH 135 ZZ-MSAMRVPAQLLGLLLLWLPGARCAVDNKFNKEQQNAFYEILHLPNLNEEQRNAFIQSLK LumicanDPPSQSANLLAEAKKLNDAQAPKVDNKFNKEQQNAFYEILHLPNLNEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPK GGGS EAHKSEIAHRYNDLGEQHFKGLVLIAFSQYLQKCSYDEHAKLVQEVTDFAKTCVADESAANCDKSLHTLFGDKLCAIPNLRENYGELADCCTKQEPERNECFLQHKDDNPSLPPFERPEAEAMCTSFKENPTTFMGHYLHEVARRHPYFYAPELLYYAEQYNEILTQCCAEADKESCLTPKLDGVKEKALVSSVRQRMKCSSMQKFGERAFKAWAVARLSQTFPNADFAEITKLATDLTKVNKECCHGDLLECADDRAELAKYMCENQATISSKLQTCCDKPLLKKAHCLSEVEHDTMPADLPAIAADFVEDQEVCKNYAEAKDVFLGTFLYEYSRRHPDYSVSLLLRLAKKYEATLEKCCAEANPPACYGTVLAEFQPLVEEPKNLVKTNCDLYEKLGEYGFQNAILVRYTQKAPQVSTPTLVEAARNLGRVGTKCCTLPEDQRLPCVEDYLSAILNRVCLLHEKTPVSEHVTKCCSGSLVERRPCFSALTVDETYVPKEFKAETFTFHSDICTLPEKEKQIKKQTALAELVKHKPKATAEQLKTVMDDFAQFLDTCCKAADKDTCFSTEGPNLVTRCKDALA GGG SGGGSQYYDYDIPLFMYGQISPNCAPECNCPHSYPTAMYCDDLKLKSVPMVPPGIKYLYLRNNQIDHIDEKAFENVTDLQWLILDHNLLENSKIKGKVFSKLKQLKKLHINYNNLTESVGPLPKSLQDLQLTNNKISKLGSFDGLVNLTFIYLQHNQLKEDAVSASLKGLKSLEYLDLSFNQMSKLPAGLPTSLLTLYLDNNKISNIPDEYFKRFTGLQYLRLSHNELADSGVPGNSFNISSLLELDLSYNKLKSIPTVNENLENYYLEVNELEKFDVKSFCKILGPLSYSKIKHLRLDGNPLTQSSLPPDMYECLRVANEITVN GGGSHHHHHH 136 Lumican-QYYDYDIPLFMYGQISPNCAPECNCPHSYPTAMYCDDLKLKSVPMVPPGIKYLYLR MSA-FcIII4CNNQIDHIDEKAFENVTDLQWLILDHNLLENSKIKGKVFSKLKQLKKLHINYNNLTESVGPLPKSLQDLQLTNNKISKLGSFDGLVNLTFIYLQHNQLKEDAVSASLKGLKSLEYLDLSFNQMSKLPAGLPTSLLTLYLDNNKISNIPDEYFKRFTGLQYLRLSHNELADSGVPGNSFNISSLLELDLSYNKLKSIPTVNENLENYYLEVNELEKFDVKSFCKILGPLSYSKIKHLRLDGNPLTQSSLPPDMYECLRVANEITVN GGGSGGGS EAHKSEIAHRYNDLGEQHFKgLVLIAFSQYLQKCSYDEHAKLVQEVTDFAKTCVADESAANCDKSLHTLFGDKLCAIPNLRENYGELADCCTKQEPERNECFLQHKDDNPSLPPFERPEAEAMCTSFKENPTIFMGHYLHEVARRHPYFYAPELLYYAEQYNEILFQCCAEADKESCLTPKLDGVKEKALVSSVRQRMKCSSMQKFGERAFKAWAVARLSQTFPNADFAEITKLATDLTKVNKECCHGDLLECADDRAELAKYMCENQATISSKLQTCCDKPLLKKAHCLSEVEHDTMPADLPAIAADFVEDQEVCKNYAEAKDVFLGTFLYEYSRRHPDYSVSLLLRLAKKYEATLEKCCAEANPPACYGTVLAEFQPLVEEPKNLVKTNCDLYEKLGEYGFQNAILVRYTQKAPQVSTPTLVEAARNLGRVGTKCCTLPEDQRLPCVEDYLSAILNRVCLLHEKTPVSEHVTKCCSGSLVERRPCFSALTVDETYVPKEFKAETFTFHSDICTLPEKEKQIKKQTALAELVKHKPKATAEQLKTVMDDFAQFLDTCCKAADKDTCFSTEGPNLVTRCKDALAGGGS CDCAWHLGELVWCTC HHHHHH 137 Lumican-MSA-Fn3QYYDYDIPLFMYGQISPNCAPECNCPHSYPTAMYCDDLKLKSVPMVPPGIKYLYLRNNQIDHIDEKAFENVTDLQWLILDHNLLENSKIKGKVFSKLKQLKKLHINYNNLTESVGPLPKSLQDLQLTNNKISKLGSFDGLVNLTFIYLQHNQLKEDAVSASLKGLKSLEYLDLSFNQMSKLPAGLPTSLLTLYLDNNKISNIPDEYFKRFTGLQYLRLSHNELADSGVPGNSFNISSLLELDLSYNKLKSIPTVNENLENYYLEVNELEKFDVKSFCKILGPLSYSKIKHLRLDGNPLTQSSLPPDMYECLRVANEITVN GGGSGGGS EAEKSEIAHRYNDLGEQHFKGLVLIAFSQYLQKCSYDEHAKLVQEVTDFAKTCVADESAANCDKSLHTLFGDKLCAIPNLRENYGELADCCTKQEPERNECFLQHKDDNPSLPPFERPEAEAMCTSFKENPTTFMGHYLHEVARRHPYFYAPELLYYAEQYNEILEQCCAEADKESCLTPKLDGVKEKALVSSVRQRMKCSSMQKFGERAFKAWAVARLSQEFPNADFAEITKLATDLTKVNKECCHGDLLECADDRAELAKYMCENQATISSKLQTCCDKPLLKKAHCLSEVEHDTMPADLPAIAADFVEDQEVCKNYAEAKDVFLGTFLYEYSRRHPDYSVSLLLRLAKKYEATLEKCCAEANPPACYGTVLAEFQPLVEEPKNLVKTNCDLYEKLGEYGFQNAILVRYTQKAPQVSTPTLVEAARNLGRVGTKCCTLPEDQRLPCVEDYLSAILNRVCLLHEKTPVSEHVTKCCSGSLVERRPCFSALTVDETYVPKEFKAETFTFHSDICTLPEKEKQIKKQTALAELVKHKPKATAEQLKTVMDDFAQFLDTCCKAADKDTCFSTEGPNLVTRCKDALA GGGS VSDVPRDLEVVAATPTSLLISWCCSDNCSNSYRITYGETGGNSPVQEFTVPRSCFMATISGLKPGVDYTITAYAVTDSNGPHPISINYRT HH HHHH 138Lumican-MSA- QYYDYDIPLFMYGQISPNCAPECNCPHSYPTAMYCDDLKLKSVPMVPPGIKYLYLRSpG2 NNQIDHIDEKAFENVTDLQWLILDHNLLENSKIKGKVFSKLKQLKKLHINYNNLTESVGPLPKSLQDLQLTNNKISKLGSFDGLVNLTFIYLQHNQLKEDAVSASLKGLKSLEYLDLSFNQMSKLPAGLPTSLLTLYLDNNKISNIPDEYFKRFTGLQYLRLSHNELADSGVPGNSFNISSLLELDLSYNKLKSIPTVNENLENYYLEVNELEKFDVKSFCKILGPLSYSKIKHLRLDGNPLTQSSLPPDMYECLRVANEITVN GGGSGGGS EAHKSEIAHRYNDLGEQHFKGLVLIAFSQYLQKCSYDEHAKLVQEVTDFAKTCVADESAANCDKSLHTLFGDKLCAIPNLRENYGELADCCTKQEPERNECFLQHKDDNPSLPPFERPEAEAMCTSFKENPTTFMGHYLHEVARRHPYFYAPELLYYAEQYNEILTQCCAEADKESCLTPKLDGVKEKALVSSVRQRMKCSSMQKFGERAFKAWAVARLSQTFPNADFAEITKLATDLTKVNKECCHGDLLECADDRAELAKYMCENQATISSKLQTCCDKPLLKKAHCLSEVEHDTMPADLPAIAADFVEDQEVCKNYAEAKDVFLGTFLYEYSRRHPDYSVSLLLRLAKKYEATLEKCCAEANPPACYGTVLAEFQPLVEEPKNLVKTNCDLYEKLGEYGFQNAILVRYTQKAPQVSTPTLVEAARNLGRVGTKCCTLPEDQRLPCVEDYLSAILNRVCLLHEKTPVSEHVTKCCSGSLVERRPCFSALTVDETYVPKEFKAETFTFHSDICTLPEKEKQIKKQTALAELVKHKPKATAEQLKTVMDDFAQFLDTCCKAADKDTCFSTEGPNLVTRCKDALA GGGS TYKLVINGKTLKGETTTEAVDAATAEKVFKQYANDYGVDGEWTYDDATKTFTVTEKPEVIDASELTPAVTTYKLVINGKTLKGETTTKAVDAETAEKAFKQYANDYGVDGVWTYDDATKTFTVTE HHHHHH 139 Lumican-MSA-QYYDYDIPLFMYGQISPNCAPECNCPHSYPTAMYCDDLKLKSVPMVPPGIKYLYLR RRGWNNQIDHIDEKAFENVTDLQWLILDHNLLENSKIKGKVFSKLKQLKKLHINYNNLTESVGPLPKSLQDLQLTNNKISKLGSFDGLVNLTFIYLQHNQLKEDAVSASLKGLKSLEYLDLSFNQMSKLPAGLPTSLLTLYLDNNKISNIPDEYFKRFTGLQYLRLSHNELADSGVPGNSFNISSLLELDLSYNKLKSIPTVNENLENYYLEVNELEKFDVKSFCKILGPLSYSKIKHLRLDGNPLTQSSLPPDMYECLRVANEITVN GGGSGGGS EAHKSEIAHRYNDLGEQHFKGLVLIAFSQYLQKCSYDEHAKLVQEVTDFAKTCVADESAANCDKSLHTLFGDKLCAIPNLRENYGELADCCTKQEPERNECFLQHKDDNPSLPPFERPEAEAMCTSFKENPTTFMGHYLHEVARRHPYFYAPELLYYAEQYNEILFQCCAEADKESCLTPKLDGVKEKALVSSVRQRMKCSSMQKFGERAFKAWAVARLSQTFPNADFAEITKLATDLTKVNKECCHGDLLECADDRAELAKYMCENQATISSKLQTCCDKPLLKKAHCLSEVEHDTMPADLPAIAADFVEDQEVCKNYAEAKDVFLGTFLYEYSRRHPDYSVSLLLRLAKKYEATLEKCCAEANPPACYGTVLAEFQPLVEEPKNLVKTNCDLYEKLGEYGFQNAILVRYTQKAPQVSTPTLVEAARNLGRVGTKCCTLPEDQRLPCVEDYLSAILNRVCLLHEKTPVSEHVTKCCSGSLVERRPCFSALTVDETYVPKEFKAETFTFHSDICTLPEKEKQIKKQTALAELVKHKPKATAEQLKTVMDDFAQFLDTCCKAADKDTCFSTEGPNLVTRCKDALA GGGS RRGW HHHHHH 140 Lumican-QYYDYDIPLFMYGQISPNCAPECNCPHSYPTAMYCDDLKLKSVPMVPPGIKYLYLR MSA-WGRRNNQIDHIDEKAFENVTDLQWLILDHNLLENSKIKGKVFSKLKQLKKLHINYNNLTESVGPLPKSLQDLQLTNNKISKLGSFDGLVNLTFIYLQHNQLKEDAVSASLKGLKSLEYLDLSFNQMSKLPAGLPTSLLTLYLDNNKISNIPDEYFKRFTGLQYLRLSHNELADSGVPGNSFNISSLLELDLSYNKLKSIPTVNENLENYYLEVNELEKFDVKSFCKILGPLSYSKIKHLRLDGNPLTQSSLPPDMYECLRVANEITVN GGGSGGGS EAHKSEIAHRYNDLGEQHFKGLVLIAFSQYLQKCSYDEHAKLVQEVTDFAKTCVADESAANCDKSLHTLFGDKLCAIPNLRENYGELADCCTKQEPERNECFLQHKDDNPSLPPFERPEAEAMCTSFKENPTTFMGHYLHEVARRHPYFYAPELLYYAEQYNEILTQCCAEADKESCLTPKLDGVKEKALVSSVRQRMKCSSMQKFGERAFKAWAVARLSQEFPNADFAEITKLATDLTKVNKECCHGDLLECADDRAELAKYMCENQATISSKLQTCCDKPLLKKAHCLSEVEHDTMPADLPAIAADFVEDQEVCKNYAEAKDVFLGTFLYEYSRRHPDYSVSLLLRLAKKYEATLEKCCAEANPPACYGTVLAEFQPLVEEPKNLVKTNCDLYEKLGEYGFQNAILVRYTQKAPQVSTPTLVEAARNLGRVGTKCCTLPEDQRLPCVEDYLSAILNRVCLLHEKTPVSEHVTKCCSGSLVERRPCFSALTVDETYVPKEFKAETFTFHSDICTLPEKEKQIKKQTALAELVKHKPKATAEQLKTVMDDFAQFLDTCCKAADKDTCFSTEGPNLVTRCKDALAGGGS WGRR HHHHHH 141 4420 LC-DVVMTQTPLSLPVSLGDQASISCRSSQSLVHSNGNTYLRWYLQKPGQSPKVLIYKV murine kappaSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYFCSQSTHVPWTFGGGTKLEIK chainRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC 142 4420 HC-DVKLDETGGGLVQPGRPMKLSCVASGFTFSDYWMNWVRQSPEKGLEWVAQIRNKPY LumicanNYETYYSDSVKGRFTISRDDSKSSVYLQMNNLRVEDMGIYYCTGSYYGMDYWGQGT (LALA-PG) SVTVSAKTTAPSVYPLAPVCGGTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPALLQSGLYTLSSSVTVTSNTWPSQTITCNVAHPASSTKVDKKIEPRVPITQNPCPPLKECPPCAAPDAAGGPSVFIFPPKIKDVLMISLSPMVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNRALGSPIEKTISKPRGPVRAPQVYVLPPPAEEMTKKEFSLTCMITGFLPAEIAVDWTSNGRTEQNYKNTATVLDSDGSYFMYSKLRVQKSTWERGSLFACSVVHEGLHNHLTTKTISR

143 4220 HC- DVKLDETGGGLVQPGRPMKLSCVASGFTFSDYWMNWVRQSPEKGLEWVAQIRNKPYmIgG2c NYETYYSDSVKGRFTISRDDSKSSVYLQMNNLRVEDMGIYYCTGSYYGMDYWGQGT SVTVSAKTTAPSVYPLAPVCGGTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPALLQSGLYTLSSSVTVTSNTWPSQTITCNVAHPASSTKVDKKIEPRVPITQNPCPPLKECPPCAAPDAAGGPSVFIFPPKIKDVLMISLSPMVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNRALGSPIEKTISKPRGPVRAPQVYVLPPPAEEMTKKEFSLTCMITGFLPAEIAVDWTSNGRTEQNYKNTATVLDSDGSYFMYSKLRVQKSTWERGSLFACSVVHEGLHNHLTTKTISR SLGK 1443/23 LC- METDTLLLWVLLLWVPGSTGDTVLTQSPALAVSPGERVTISCRASESVSTRMHWYQmurine kappa QRPGQPPKLLIYVASRLESGVPARFSGGGSGTDFTLTIDPVEANDTATYFCQQSWNDPWTFGGGTKLELK RADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPI VKSFNRNEC 1453/23 HC- MDIWLSLVFLVLFIKGVQCEVQLVESGGGLVQPGRSLKLSCAASGFTLSDYYMAWVlumican RQAPKKGLEWVASINYEGSSTYYGESVKGRFTISRDNAKSTLYLQMNSLRSEDTATYYCVRHDNYFDYWGQGVLVTVSS AKTTAPSVYPLAPVCGGTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPALLQSGLYTLSSSVTVTSNTWPSQTITCNVAHPASSTKVDKKIEPRVPITQNPCPPLKECPPCAAPDAAGGPSVFIFPPKIKDVLMISLSPMVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNRALGSPlEKTISKPRGPVRAPQVYVLPPPAEEMTKKEFSLTCMITGFLPAEIAVDWTSNGRTEQNYKNTATVLDSDGSYFMYSKLRVQKSTWERGSLFAC

146  LOB12.3 LC-DIQMTQSPASLSASLEEIVTITCQASQDIGNWLAWYHQKPGKSPQLLIYGSTSLAD murine kappaGVPSRFSGSSSGSQYSLKISRLQVEDIGIYYCLQAYGAPWTFGGGTKLELK RADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC 147 LOB12.3 HC-DVQLVESGGGLVQPGRSLKLSCAASGFIFSYFDMAWVRQAPTKGLEWVASISPDGS LumicanIPYYRDSVKGRFTVSRENAKSSLYLQMDSLRSEDTATYYCARRSYGGYSEIDYWGQ GVMVTVSSAKTTAPSVYPLAPVCGGTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPALLQSGLYTLSSSVTVTSNTWPSQTITCNVAHPASSTKVDKKIEPRVPITQNPCPPLKECPPCAAPDAAGGPSVFIFPPKIKDVLMISLSPMVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNRALGSPIEKTISKPRGPVRAPQVYVLPPPAEEMTKKEFSLTCMITGFLPAEIAVDWTSNGRTEQNYKNTATVLDSDGSYFMYSKLRVQKSTWERGSLFACSVVHEGLHNHLTTKT

148 OX86 LC- DIVMTQGALPNPVPSGESASITCRSSQSLVYKDGQTYLNWFLQRPGQSPQLLTYWMmurine kappa STRASGVSDRFSGSGSGTYFTLKISRVRAEDAGVYYCQQVREYPFTFGSGTKLEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC 149 OX86 HC-QVQLKESGPGLVQPSQTLSLTCTVSGFSLTGYNLHWVRQPPGKGLEWMGRMRYDGD LumicanTYYNSVLKSRLSISRDTSKNQVFLKMNSLQTDDTAIYYCTRDGRGDSFDYWGQGVM VTVSSAKTTAPSVYPLAPVCGGTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPALLQSGLYTLSSSVTVTSNTWPSQTITCNVAHPASSTKVDKKIEPRVPITQNPCPPLKECPPCAAPDAAGGPSVFIFPPKIKDVLMISLSPMVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNRALGSPIEKTISKPRGPVRAPQVYVLPPPAEEMTKKEFSLTCMITGFLPAEIAVDWTSNGRTEQNYKNTATVLDSDGSYFMYSKLRVQKSTWERGSLFACSVVHEGLHNHLTTKTISR

150 2C11 LC- DIQMTQSPSSLPASLGDRVTINCQASQDISNYLNWYQQKPGKAPKLLIYYTNKLADmurine kappa GVPSRFSGSGSGRDSSFTISSLESEDIGSYYCQQYYNYPWTFGPGTKLEIKR RADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC 151 2C11 HC-EVQLVESGGGLVQPGKSLKLSCEASGFTFSGYGMHWVRQAPGRGLESVAYITSSSI lumicanNIKYADAVKGRFTVSRDNAKNLLFLQMNILKSEDTAMYYCARFDWDKNYWGQGTMV TVSSAKTTAPSVYPLAPVCGGTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPALLQSGLYTLSSSVTVTSNTWPSQTITCNVAHPASSTKVDKKIEPRVPITQNPCPPLKECPPCAAPDAAGGPSVFIFPPKIKDVLMISLSPMVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNRALGSPIEKTISKPRGPVRAPQVYVLPPPAEEMTKKEFSLTCMITGFLPAEIAVDWTSNGRTEQNYKNTATVLDSDGSYFMYSKLRVQKSTWERGSLFACSVVHEGLHNHLTTKTISRS

152 T2A peptide

(furin cleavage RRKRGSGEGRGSLLTCGDVEENPGP site-GSG-T2A) 153 CCL3-APYGADTPTACCFSYSRKIPRQFIVDYFETSSLCSQPGVIFLTKRNRQICADSKET LumicanWVQEYITDLELNA GGGSGGGSGGGS QYYDYDIPLFMYGQISPNCAPECNCPHSYPTAMYCDDLKLKSVPMVPPGIKYLYLRNNQIDHIDEKAFENVTDLQWLILDHNLLENSKIKGKVFSKLKQLKKLHINYNNLTESVGPLPKSLQDLQLTNNKISKLGSFDGLVNLTFIYLQHNQLKEDAVSASLKGLKSLEYLDLSFNQMSKLPAGLPTSLLTLYLDNNKISNIPDEYFKRFTGLQYLRLSHNELADSGVPGNSFNISSLLELDLSYNKLKSIPTVNENLENYYLEVNELEKFDVKSFCKILGPLSYSKIKHLRLDGNPLTQSSLPPDMYECLRVANEITVNGGGSHHHHHH 154 Lumican-QYYDYDIPLFMYGQISPNCAPECNCPHSYPTAMYCDDLKLKSVPMVPPGIKYLYLR CCL3NNQIDHIDEKAFENVTDLQWLILDHNLLENSKIKGKVFSKLKQLKKLHINYNNLTESVGPLPKSLQDLQLTNNKISKLGSFDGLVNLTFIYLQHNQLKEDAVSASLKGLKSLEYLDLSFNQMSKLPAGLPTSLLTLYLDNNKISNIPDEYFKRFTGLQYLRLSHNELADSGVPGNSFNISSLLELDLSYNKLKSIPTVNENLENYYLEVNELEKFDVKSFCKILGPLSYSKIKHLRLDGNPLTQSSLPPDMYECLRVANEITVN GGGSGGGSGGGS APYGADTPTACCFSYSRKIPRQFIVDYFETSSLCSQPGVIFLTKRNRQICADSKETWVQEYITDLELNAGGGSHHHHHH 155 CCL3APYGADTPTACCFSYSRKIPRQFIVDYFETSSLCSQPGVIFLTKRNRQICADSKET WVQEYITDLELNAGGGSHHHHHH 156 CCL4-APMGSDPPTSCCFSYTSRQLHRSFVMDYYETSSLCSKPAVVFLTKRGRQICANPSE lumicanPWVTEYMSDLELN GGGSGGGSGGGS QYYDYDIPLFMYGQISPNCAPECNCPHSYPTAMYCDDLKLKSVPMVPPGIKYLYLRNNQIDHIDEKAFENVTDLQWLILDHNLLENSKIKGKVFSKLKQLKKLHINYNNLTESVGPLPKSLQDLQLTNNKISKLGSFDGLVNLTFIYLQHNQLKEDAVSASLKGLKSLEYLDLSFNQMSKLPAGLPTSLLTLYLDNNKISNIPDEYFKRFTGLQYLRLSHNELADSGVPGNSFNISSLLELDLSYNKLKSIPTVNENLENYYLEVNELEKFDVKSFCKILGPLSYSKIKHLRLDGNPLTQSSLPPDMYECL RVANEITVNGGGSHHHHHH 157 Lumican-QYYDYDIPLFMYGQISPNCAPECNCPHSYPTAMYCDDLKLKSVPMVPPGIKYLYLR CCL4NNQIDHIDEKAFENVTDLQWLILDHNLLENSKIKGKVFSKLKQLKKLHINYNNLTESVGPLPKSLQDLQLTNNKISKLGSFDGLVNLTFIYLQHNQLKEDAVSASLKGLKSLEYLDLSFNQMSKLPAGLPTSLLTLYLDNNKISNIPDEYFKRFTGLQYLRLSHNELADSGVPGNSFNISSLLELDLSYNKLKSIPTVNENLENYYLEVNELEKFDVKSFCKILGPLSYSKIKHLRLDGNPLTQSSLPPDMYECLRVANEITVN GGGSGGGSGGGS APMGSDPPTSCCFSYTSRQLHRSFVMDYYETSSLCSKPAVVFLTKRGRQICANPSEPWVT EYMSDLELNGGGSHHHHHH 158 CCL4APMGSDPPTSCCFSYTSRQLHRSFVMDYYETSSLCSKPAVVFLTKRGRQICANPSE PWVTEYMSDLELNGGGSHHHHHH 159 CCL5-SPYGSDTTPCCFAYLSLALPRAHVKEYFYTSSKCSNLAVVFVTRRNRQVCANPEKK lumicanWVQEYINYLEMS GGGSGGGSGGGS QYYDYDIPLFMYGQISPNCAPECNCPHSYPTAMYCDDLKLKSVPMVPPGIKYLYLRNNQIDHIDEKAFENVTDLQWLILDHNLLENSKIKGKVFSKLKQLKKLHINYNNLTESVGPLPKSLQDLQLTNNKISKLGSFDGLVNLTFIYLQHNQLKEDAVSASLKGLKSLEYLDLSFNQMSKLPAGLPTSLLTLYLDNNKISNIPDEYFKRFTGLQYLRLSHNELADSGVPGNSFNISSLLELDLSYNKLKSIPTVNENLENYYLEVNELEKFDVKSFCKILGPLSYSKIKHLRLDGNPLTQSSLPPDMYECLR VANEITVNGGGSHHHHHH 160 Lumican-QYYDYDIPLFMYGQISPNCAPECNCPHSYPTAMYCDDLKLKSVPMVPPGIKYLYLR CCL5NNQIPHIDEKAFENVTDLQWLILDHNLLENSKIKGKVFSKLKQLKKLHINYNNLTESVGPLPKSLQDLQLTNNKISKLGSFDGLVNLTFIYLQHNQLKEDAVSASLKGLKSLEYLDLSFNQMSKLPAGLPTSLLTLYLDNNKISNIPDEYFKRFTGLQYLRLSHNELADSGVPGNSFNISSLLELDLSYNKLKSIPTVNENLENYYLEVNELEKFDVKSFCKILGPLSYSKIKHLRLDGNPLTQSSLPPDMYECLRVANEITVN GGGSGGGSGGGS SPYGSDTTPCCFAYLSLALPRAHVKEYFYTSSKCSNLAVVFVTRRNRQVCANPEKKWVQE YINYLEMSGGGSHHHHHH 161 CCL5SPYGSDTTPCCFAYLSLALPRAHVKEYFYTSSKCSNLAWFVTRRNRQVCANPEKK WVQEYINYLEMSGGGSHHHHHH 162 CCL19-GANDAEDCCLSVTQRPIPGNIVKAFRYLLNEDGCRVPAVVFTTLRGYQLCAPPDQP LumicanWVDRIIRRLKKSSAKNKGNSTRRSPVS GGGSGGGSGGGS QYYDYDIPLFMYGQISPNCAPECNCPHSYPTAMYCDDLKLKSVPMVPPGIKYLYLRNNQIDHIDEKAFENVTDLQWLILDHNLLENSKIKGKVFSKLKQLKKLHINYNNLTESVGPLPKSLQDLQLTNNKISKLGSFDGLVNLTFIYLQHNQLKEDAVSASLKGLKSLEYLDLSFNQMSKLPAGLPTSLLTLYLDNNKISNIPDEYFKRFTGLQYLRLSHNELADSGVPGNSFNISSLLELDLSYNKLKSIPTVNENLENYYLEVNELEKFDVKSFCKILGPLSYSKIKHLRLDGNPLTQSSLPPDMYECLRVANEITVN GGGSHHHHHH 163 Lumican-QYYDYDIPLFMYGQISPNCAPECNCPHSYPTAMYCDDLKLKSVPMVPPGIKYLYLR CCL19NNQIPHIDEKAFENVTDLQWLILDHNLLENSKIKGKVFSKLKQLKKLHINYNNLTESVGPLPKSLQDLQLTNNKISKLGSFDGLVNLTFIYLQHNQLKEDAVSASLKGLKSLEYLDLSFNQMSKLPAGLPTSLLTLYLDNNKISNIPDEYFKRFTGLQYLRLSHNELADSGVPGNSFNISSLLELDLSYNKLKSIPTVNENLENYYLEVNELEKFDVKSFCKILGPLSYSKIKHLRLDGNPLTQSSLPPDMYECLRVANEITVN GGGSGGGSGGG GANDAEDCCLSVTQRPIPGNIVKAFRYLLNEDGCRVPAVVFTTLRGYQLCAPPDQPWVDRIIRRLKKSSAKNKGNSTRRSPVS GGGSHHHHHH 164 CCL19GANDAEDCCLSVTQRPIPGNIVKAFRYLLNEDGCRVPAVVFTTLRGYQLCAPPDQPWVDRIIRRLKKSSAKNKGNSTRRSPVS GGGSHHHHHH 165 CCL21c-SDGGGQDCCLKYSQKKIPYSIVRGYRKQEPSLGCPIPAILFLPRKHSKPELCANPE LumicanEGWVQNLMRRLDQPPAPGKQSPGCRKNRGTSKSGKKGKGSKGCKRTEQTQPSRG GG GSGGGSGGGSQYYDYDIPLFMYGQISPNCAPECNCPHSYPTAMYCDDLKLKSVPMVPPGIKYLYLRNNQIDHIDEKAFENVTDLQWLILDHNLLENSKIKGKVFSKLKQLKKLHINYNNLTESVGPLPKSLQDLQLTNNKISKLGSFDGLVNLTFIYLQHNQLKEDAVSASLKGLKSLEYLDLSFNQMSKLPAGLPTSLLTLYLDNNKISNIPDEYFKRFTGLQYLRLSHNELADSGVPGNSFNISSLLELDLSYNKLKSIPTVNENLENYYLEVNELEKFDVKSFCKILGPLSYSKIKHLRLDGNPLTQSSLPPDMYECLRVANEITVN GGGSHH HHHH 166Lumican- QYYDYDIPLFMYGQISPNCAPECNCPHSYPTAMYCDDLKLKSVPMVPPGIKYLYLR CCL21cNNQIPHIDEKAFENVTDLQWLILDHNLLENSKIKGKVFSKLKQLKKLHINYNNLTESVGPLPKSLQDLQLTNNKISKLGSFDGLVNLTFIYLQHNQLKEDAVSASLKGLKSLEYLDLSFNQMSKLPAGLPTSLLTLYLDNNKISNIPDEYFKRFTGLQYLRLSHNELADSGVPGNSFNISSLLELDLSYNKLKSIPTVNENLENYYLEVNELEKFDVKSFCKILGPLSYSKIKHLRLDGNPLTQSSLPPDMYECLRVANEITVN GGGSGGGSGGG SDGGGQDCCLKYSQKKIPYSIVRGYRKQEPSLGCPIPAILFLPRKHSKPELCANPEEGWVQNLMRRLDQPPAPGKQSPGCRKNRGTSKSGKKGKGSKGCKRTEQTQPSRG GGGSHHH HHH 167 CCL21cSDGGGQDCCLKYSQKKIPYSIVRGYRKQEPSLGCPIPAILFLPRKHSKPELCANPEEGWVQNLMRRLDQPPAPGKQSPGCRKNRGTSKSGKKGKGSKGCKRTEQTQPSRG GG GSHHHHHH 168truncated SDGGGQDCCLKYSQKKIPYSIVRGYRKQEPSLGCPIPAILFLPRKHSKPELCANPECCL21c- EGWVQNLMRRLDQPPAPGK GGGSGGGSGGGS QYYDYDIPLFMYGQISPNCAPECNCLumican PHSYPTAMYCDDLKLKSVPMVPPGIKYLYLRNNQIDHIDEKAFENVTDLQWLILDHNLLENSKIKGKVFSKLKQLKKLHINYNNLTESVGPLPKSLQDLQLTNNKISKLGSFDGLVNLTFIYLQHNQLKEDAVSASLKGLKSLEYLDLSFNQMSKLPAGLPTSLLTLYLDNNKISNIPDEYFKRFTGLQYLRLSHNELADSGVPGNSFNISSLLELDLSYNKLKSIPTVNENLENYYLEVNELEKFDVKSFCKILGPLSYSKIKHLRLDGNPLTQSSLPP DMYECLRVANEITVNGGGSHHHHHH 169 Lumican-QYYDYDIPLFMYGQISPNCAPECNCPHSYPTAMYCDDLKLKSVPMVPPGIKYLYLR truncatedNNQIDHIDEKAFENVTDLQWLILDHNLLENSKIKGKVFSKLKQLKKLHINYNNLTE CCL21cSVGPLPKSLQDLQLTNNKISKLGSFDGLVNLTFIYLQHNQLKEDAVSASLKGLKSLEYLDLSFNQMSKLPAGLPTSLLTLYLDNNKISNIPDEYFKRFTGLQYLRLSHNELADSGVPGNSFNISSLLELDLSYNKLKSIPTVNENLENYYLEVNELEKFDVKSFCKILGPLSYSKIKHLRLDGNPLTQSSLPPDMYECLRVANEITVN GGGSGGGSGGGS SDGGGQDCCLKYSQKKIPYSIVRGYRKQEPSLGCPIPAILFLPRKHSKPELCANPEEGWV QNLMRRLDQPPAPGKGGGSHHHHHH 170 truncatedSDGGGQDCCLKYSQKKIPYSIVRGYRKQEPSLGCPIPAILFLPRKHSKPELCANPE CCL21cEGWVQNLMRRLDQPPAPGK GGGSHHHHHH 171 CCL11HPGSIPTSCCFIMTSKKIPNTLLKSYKRITNNRCTLKAIVFKTRLGKEICADPKKKWVQDATKHLDQKLQTPKP GGGSHHHHHH 172 CCL11-HPGSIPTSCCFIMTSKKIPNTLLKSYKRITNNRCTLKAIVFKTRLGKEICADPKKK LumicanWVQDATKHLDQKLQTPKP GGGSGGGSGGGS QYYDYDIPLFMYGQISPNCAPECNCPHSYPTAMYCDDLKLKSVPMVPPGIKYLYLRNNQIDHIDEKAFENVTDLQWLILDHNLLENSKIKGKVFSKLKQLKKLHINYNNLTESVGPLPKSLQDLQLTNNKISKLGSFDGLVNLTFIYLQHNQLKEDAVSASLKGLKSLEYLDLSFNQMSKLPAGLPTSLLTLYLDNNKISNIPDEYFKRFTGLQYLRLSHNELADSGVPGNSFNISSLLELDLSYNKLKSIPTVNENLENYYLEVNELEKFDVKSFCKILGPLSYSKIKHLRLDGNPLTQSSLPPD MYECLRVANEITVNGGGSHHHHHH 173 CLEC2-QQKYLLAEKENLSATLQQLAKKFCQELIRQSEIKTKSTFEHKCSPCATKWRYHGDS MSA-lumicanCYGFFRRNLTWEESKQYCTEQNATLVKTASQSTLDYIAERITSVRWIGLSRQNSKKDWMWEDSSVLRKNGINLSGNTEENMNCAYLHNGKIHPASCKERHYLICERNAGMTRVDQLLGGGGGGGGGGGGEAHKSEIAHRYNDLGEQHFKGLVLIAFSQYLQKCSYDEHAKLVQEVTDFAKTCVADESAANCDKSLHTLFGDKLCAIPNLRENYGELADCCTKQEPERNECFLQHKDDNPSLPPFERPEAEAMCTSFKENPTTFMGHYLHEVARRHPYFYAPELLYYAEQYNEI-TQCCAEADKESC-TPKLDGVKEKALVSSVRQRMKCSSMQKFGERAFKAWAVARLSQTFPNADFAEITKLATDLTKVNKECCHGDLLECADDRAELAKYMCENQATISSKLQTCCDKPLLKKAHCLSEVEHDTMPADLPAIAADFVEDQEVCKNYAEAKDVFLGTFLYEYSRRHPDYSVSLLLRLAKKYEATLEKCCAEANPPACYGTVLAEFQPLVEEPKNLVKTNCDLYEKLGEYGFQNAILVRYTQKAPQVSTPTLVEAARNLGRVGTKCCTLPEDQRLPCVEDYLSAILNRVCLLHEKTPVSEHVTKCCSGSLVERRPCFSALTVDETYVPKEFKAETFTFHSDICTLPEKEKQIKKQTALAELVKHKPKATAEQ

174 CLEC2-MSA QQKYLLAEKENLSATLQQLAKKFCQELIRQSEIKTKSTFEHKCSPCATKWRYHGDSCYGFFRRNLTWEESKQYCTEQNATLVKTASQSTLDYIAERITSVRWIGLSRQNSKKDWMWEDSSVLRKNGINLSGNTEENMNCAYLHNGKIHPASCKERHYLICERNAGMTRVDQLLGGGSGGGSGGGSEAHKSEIAHRYNDLGEQHFKGLVLIAFSQYLQKCSYDEHAKLVQEVTDFAKTCVADESAANCDKSLHTLFGDKLCAIPNLRENYGELADCCTKQEPERNECFLQHKDDNPSLPPFERPEAEAMCTSFKENPTTFMGHYLHEVARRHPYFYAPELLYYAEQYNEILTQCCAEADKESCLTPKLDGVKEKALVSSVRQRMKCSSMQKFGERAFKAWAVARLSQTFPNADFAEITKLATDLTKVNKECCHGDLLECADDRAELAKYMCENQATISSKLQTCCDKPLLKKAHCLSEVEHDTMPADLPAIAADFVEDQEVCKNYAEAKDVFLGTFLYEYSRRHPDYSVSLLLRLAKKYEATLEKCCAEANPPACYGTVLAEFQPLVEEPKNLVKTNCDLYEKLGEYGFQNAILVRYTQKAPQVSTPTLVEAARNLGRVGTKCCTLPEDQRLPCVEDYLSAILNRVCLLHEKTPVSEHVTKCCSGSLVERRPCFSALTVDETYVPKEFKAETFTFHSDICTLPEKEKQIKKQTALAELVKHKPKATAEQLKTVMDDFAQFLDTCCKAADKDTCFSTEGPNLVTRCKDALAHHHHHH 175 IFNg-MSA-HGTVIESLESLNNYFNSSGIDVEEKSLFLDIWRNWQKDGDMKILQSQIISFYLRLF lumicanEVLKDNQAISNNISVIESHLITTFFSNSKAKKDAFMSIAKFEVNNPQVQRQAFNELIRVVHQLLPESSLRKRKRSRC GSGGGS EAHKSEIAHRYNDLGEQHFKGLVLIAFSQYLQKCSYDEHAKLVQEVTDFAKTCVADESAANCDKSLHTLFGDKLCAIPNLRENYGELADCCTKQEPERNECFLQHKDDNPSLPPFERPEAEAMCTSFKENPTTFMGHYLHEVARRHPYFYAPELLYYAEQYNEILTQCCAEADKESCLTPKLDGVKEKALVSSVRQRMKCSSMQKFGERAFKAWAVARLSQTFPNADFAEITKLATDLTKVNKECCHGDLLECADDRAELAKYMCENQATISSKLQTCCDKPLLKKAHCLSEVEHDTMPADLPAIAADFVEDQEVCKNYAEAKDVFLGTFLYEYSRRHPDYSVSLLLRLAKKYEATLEKCCAEANPPACYGTVLAEFQPLVEEPKNLVKTNCDLYEKLGEYGFQNAILVRYTQKAPQVSTPTLVEAARNLGRVGTKCCTLPEDQRLPCVEDYLSAILNRVCLLHEKTPVSEHVTKCCSGSLVERRPCFSALTVDETYVPKEFKAETFTFHSDICTLPEKEKQIKKQTALAELVKHKPKATAEQLKTVMDDFAQFLDTCCKAADKDTCFSTEGPNLVTRCKDALA GGGSG

176 IFNg-IFNg- HGTVIESLESLNNYFNSSGIDVEEKSLFLDIWRNWQKDGDMKILQSQIISFYLRLFMSA-lumican EVLKDNQAISNNISVIESHLITTFFSNSKAKKDAFMSIAKFEVNNPQVQRQAFNELIRVVHQLLPESSLRKRKRSRC GGGSGGGSGGGSGGGS HGTVIESLESLNNYFNSSGIDVEEKSLFLDIWRNWQKDGDMKILQSQIISFYLRLFEVLKDNQAISNNISVIESHLITTFFSNSKAKKDAFMSIAKFEVNNPQVQRQAFNELIRVVHQLLPESSLRKRKRS RC GSGGGSEAHKSEIAHRYNDLGEQHFKGLVLIAFSQYLQKCSYDEHAKLVQEVTDFAKTCVADESAANCDKSLHTLFGDKLCAIPNLRENYGELADCCTKQEPERNECFLQHKDDNPSLPPFERPEAEAMCTSFKENPTTFMGHYLHEVARRHPYFYAPELLYYAEQYNEILTQCCAEADKESCLTPKLDGVKEKALVSSVRQRMKCSSMQKFGERAFKAWAVARLSQTFPNADFAEITKLATDLTKVNKECCHGDLLECADDRAELAKYMCENQATISSKLQTCCDKPLLKKAHCLSEVEHDTMPADLPAIAADFVEDQEVCKNYAEAKDVFLGTFLYEYSRRHPDYSVSLLLRLAKKYEATLEKCCAEANPPACYGTVLAEFQPLVEEPKNLVKTNCDLYEKLGEYGFQNAILVRYTQKAPQVSTPTLVEAARNLGRVGTKCCTLPEDQRLPCVEDYLSAILNRVCLLHEKTPVSEHVTKCCSGSLVERRPCFSALTVDETYVPKEFKAETFTFHSDICTLPEKEKQIKKQTALAELVKHKPKATAEQLKTVMDDFA

177 IFNg-IFNg- HGTVTESLESLNNYFNSSGIDVEEKSLFLDIWRNWQKDGDMKILQSQIISFYLRLFMSA EVLKDNQAISNNISVIESHLITTFFSNSKAKKDAFMSIAKFEVNNPQVQRQAFNELIRVVHQLLPESSLRKRKRSRC GGGSGGGSGGGSGGGS HGTVIESLESLNNYFNSSGIDVEEKSLFLDIWRNWQKDGDMKILQSQIISFYLRLFEVLKDNQAISNNISVIESHLITTFFSNSKAKKDAFMSIAKFEVNNPQVQRQAFNELIRVVHQLLPESSLRKRKRS RC GSGGGSEAHKSEIAHRYNDLGEQHFKGLVLIAFSQYLQKCSYDEHAKLVQEVTDFAKTCVADESAANCDKSLHTLFGDKLCAIPNLRENYGELADCCTKQEPERNECFLQHKDDNPSLPPFERPEAEAMCTSFKENPTTFMGHYLHEVARRHPYFYAPELLYYAEQYNEILTQCCAEADKESCLTPKLDGVKEKALVSSVRQRMKCSSMQKFGERAFKAWAVARLSQTFPNADFAEITKLATDLTKVNKECCHGDLLECADDRAELAKYMCENQATISSKLQTCCDKPLLKKAHCLSEVEHDTMPADLPAIAADFVEDQEVCKNYAEAKDVFLGTFLYEYSRRHPDYSVSLLLRLAKKYEATLEKCCAEANPPACYGTVLAEFQPLVEEPKNLVKTNCDLYEKLGEYGFQNAILVRYTQKAPQVSTPTLVEAARNLGRVGTKCCTLPEDQRLPCVEDYLSAILNRVCLLHEKTPVSEHVTKCCSGSLVERRPCFSALTVDETYVPKEFKAETFTFHSDICTLPEKEKQIKKQTALAELVKHKPKATAEQLKTVMDDFAQFLDTCCKAADKDTCFSTEGPNLVTRCKDALAHHHHHH 178 Lumican- MSA-IFNg

HRYNDLGEQHFKGLVLIAFSQYLQKCSYDEHAKLVQEVTDFAKTCVADESAANCDKSLHTLFGDKLCAIPNLRENYGELADCCTKQEPERNECFLQHKDDNPSLPPFERPEAEAMCTSFKENPTTFMGHYLHEVARRHPYFYAPELLYYAEQYNEILTQCCAEADKESCLTPKLDGVKEKALVSSVRQRMKCSSMQKFGERAFKAWAVARLSQTFPNADFAEITKLATDLTKVNKECCHGDLLECADDRAELAKYMCENQATISSKLQTCCDKPLLKKAHCLSEVEHDTMPADLPAIAADFVEDQEVCKNYAEAKDVFLGTFLYEYSRRHPDYSVSLLLRLAKKYEATLEKCCAEANPPACYGTVLAEFQPLVEEPKNLVKTNCDLYEKLGEYGFQNAILVRYTQKAPQVSTPTLVEAARNLGRVGTKCCTLPKDQRLPCVEDYLSAILNRVCLLHEKTPVSEHVTKCCSGSLVERRPCFSALTVDETYVPKEFKAETFTFHSDICTLPEKEKQIKKQTALAELVKHKPKATAEQLKTVMDDFAQFLDTCCKAADKDTCFSTEGPNLVTRCKDALA GGGS HGTVIESLESLNNYFNSSGIDVEEKSLFLDIWRNWQKDGDMKILQSQIISFYLRLFEVLKDNQAISNNISVIESHLITTFFSNSKAKKDAFMSIAKFEVNNPQVQRQAFNELIRWHQLLPESSLRKRKRSRCHHHHHH 179 Lumican- MSA-IFNg-IFNg

HRYNDLGEQHFKGLVLIAFSQYLQKCSYDEHAKLVQEVTDFAKTCVADESAANCDKSLHTLFGDKLCAIPNLRENYGELADCCTKQEPERNECFLQHKDDNPSLPPFERPEAEAMCTSFKENPTTFMGHYLHEVARRHPYFYAPELLYYAEQYNEILTQCCAEADKESCLTPKLDGVKEKALVSSVRQRMKCSSMQKFGERAFKAWAVARLSQTFPNADFAEITKLATDLTKVNKECCHGDLLECADDRAELAKYMCENQATISSKLQTCCDKPLLKKAHCLSEVEHDTMPADLPAIAADFVEDQEVCKNYAEAKDVFLGTFLYEYSRRHPDYSVSLLLRLAKKYEATLEKCCAEANPPACYGTVLAEFQPLVEEPKNLVKTNCDLYEKLGEYGFQNAILVRYTQKAPQVSTPTLVEAARNLGRVGTKCCTLPEDQRLPCVEDYLSAILNRVCLLHEKTPVSEHVTKCCSGSLVERRPCFSALTVDETYVPKEFKAETFTFHSDICTLPEKEKQIKKQTALAELVKHKPKATAEQLKTVMDDFAQFLDTCCKAADKDTCFSTEGPNLVTRCKDALA GGGS HGTVIESLESLNNYFNSSGIDVEEKSLFLDIWRNWQKDGDMKILQSQIISFYLRLFEVLKDNQAISNNISVIESHLITTFFSNSKAKKDAFMSIAKFEVNNPQVQRQAFNELIRVVHQLLPESSLRKRKRSRC GGGSGGGSGGGSGGG SHGTVIESLESLNNYFNSSGIDVEEKSLFLDIWRNWQKDGDMKILQSQIISFYLRLFEVLKDNQAISNNISVIESHLITTFFSNSKAKKDAFMSIAKFEVNNPQVQRQAFNELIRVVHQLLPESSLRKRKRSRCHHHHHH 180 MSA-IFNg-EAHKSEIAHRYNDLGEQHFKGLVLIAFSQYLQKCSYDEHAKLVQEVTDFAKTCVAD IFNgESAANCDKSLHTLFGDKLCAIPNLRENYGELADCCTKQEPERNECFLQHKDDNPSLPPFERPEAEAMCTSFKENPYTFMGHYLHEVARRHPYFYAPKLLYYAFQYNEILTQCCAEADKESCLTPKLDGVKEKALVSSVRQRMKCSSMQKFGERAFKAWAVARLSQTFPNADFAEITKLATDLTKVNKECCHGDLLECADDRAELAKYMCENQATISSKLQTCCDKPLLKKAHCLSEVEHDTMPADLPAIAADFVEDQEVCKNYAEAKDVFLGTFLYEYSRRHPDYSVSLLLRLAKKYEATLEKCCAEANPPACYGTVLAEFQPLVEEPKNLVKTNCDLYEKLGEYGFQNAILVRYTQKAPQVSTPTLVEAARNLGRVGTKCCTLPEDQRLPCVEDYLSAILNRVCLLHEKTPVSEHVTKCCSGSLVERRPCFSALTVDETYVPKEFKAETFTFHSDICTLPEKEKQIKKQTALAELVKHKPKATAEQLKTVMDDFAQFLDTCCKAADKDTCFSTEGPNLVTRCKDALA GGGS HGTVIESLESLNNYFNSSGIDVEEKSLFLDIWRNWQKDGDMKILQSQIISFYLRLFEVLKDNQAISNNISVIESHLITTFFSNSKAKKDAFMSIAKFEVNNPQVQRQAFNELIRVVHQLLPESSLRKRKRSRC GGGSGGG SGGGSGGGSHGTVIESLESLNNYFNSSGIDVEEKSLFLDIWRNWQKDGDMKILQSQIISFYLRLFEVLKDNQAISNNISVIESHLITTFFSNSKAKKDAFMSIAKFEVNNPQVQRQAFNELIRVVHQLLPESSLRKRKRSRCHHHHHH 181 LAIR-(H)₆QEGSLPDITIFPNSSLMISQGTFVTVVCSYSDKHDLYNMVRLEKDGSTFMEKSTEPYKTEDEFEIGPVNETITGHYSCIYSKGITWSERSKTLELKVIKENVIQTPAPGPTSDTSWLKTYSIYHHHHHH 182 LumicanYYDYDIPLFMYGQISPNCAPECNCPHSYPTAMYCDDLKLKSVPMVPPGIKYLYLRN (murine)NQIDHIDEKAFENVTDLQWLILDHNLLENSKIKGKVFSKLKQLKKLHINYNNLTESVGPLPKSLQDLQLTNNKISKLGSFDGLVNLTFIYLQHNQLKEDAVSASLKGLKSLEYLDLSFNQMSKLPAGLPTSLLTLYLDNNKISNIPDEYFKRFTGLQYLRLSHNELADSGVPGNSFNISSLLELDLSYNKLKSIPTVNENLENYYLEVNELEKFDVKSFCKILGPLSYSKIKHLRLDGNPLTQSSLPPDMYECLRVANEITVN 183 murine MSAEAHKSEIAHRYNDLGEQHFKGLVLIAFSQYLQKCSYDEHAKLVQEVTDFAKTCVADESAANCDKSLHTLFGDKLCAIPNLRENYGELADCCTKQEPERNECFLQHKDDNPSLPPFERPEAEAMCTSFKENPTTFMGHYLHEVARRHPYFYAPELLYYAEQYNEILTQCCAEADKESCLTPKLDGVKEKALVSSVRQRMKCSSMQKFGERAFKAWAVARLSQTFPNADFAEITKLATDLTKVNKECCHGDLLECADDRAELAKYMCENQATISSKLQTCCDKPLLKKAHCLSEVEHDTMPADLPAIAADFVEDQEVCKNYAEAKDVFLGTFLYEYSRRHPDYSVSLLLRLAKKYEATLEKCCAEANPPACYGTVLAEFQPLVEEPKNLVKTNCDLYEKLGEYGFQNAILVRYTQKAPQVSTPTLVEAARNLGRVGTKCCTLPEDQRLPCVEDYLSAILNRVCLLHEKTPVSEHVTKCCSGSLVERRPCFSALTVDETYVPKEFKAETFTFHSDICTLPEKEKQIKKQTALAELVKHKPKATAEQLKTVMDDFAQFLDTCCKAADKDTCFSTEGPNLVTRCKDALA 184 3/23 LC-

murine kappa DTVLTQSPALAVSPGERVTISCRASESVSTRMHWYQQRPGQPPKLLIYVASRLESG(3/23 LC bold; VPARFSGGGSGTDFTLTIDPVEANDTATYFCQQSWNDPWTFGGGTKLELK RADAAPmurine kappa TVSIFPPSSEQLTSGGASVVGFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKchain DSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC underlined) 1853/23 HC- EVQLVESGGGLVQPGRSLKLSCAASGFTLSDYYMAWVRQAPKKGLEWVASINYEGSlumican (LALA- STYYGESVKGRFTISRDNAKSTLYLQMNSLRSEDTATYYCVRHDNYFDYWGQGVLVPG) (3/23 HC TVSS AKTTAPSVYPLAPVCGGTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTbold; mIgG2c FPALLQSGLYTLSSSVTVTSNTWPSQTITCNVAHPASSTKVDKKIEPRVPITQNPCunderline; PPLKECPPCAAPDAAGGPSVF1FPPKIKDVLMISLSPMVTCVVVDVSEDDPDVQISLALA-PG WFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNRALGSPIsilencing EKTISKPRGPVRAPQVYVLPPPAEEMTKKEFSLTCMITGFLPAEIAVDWTSNGRTEmutation bold QNYKNTATVLDSDGSYFMYSKLRVQKSTWERGSLFACSVVHEGLHNHLTTKTISRSunderline; Lumican dotted underline)

186 LAIR-MSA-IL2 QEGSLPDITIFPNSSLMISQGTFVTVVCSYSDKHDLYNMVRLEKDGSTFMEKSTEP (LAIR bold;-YKTEDEFEIGPVNETITGHYSCIYSKGITWSERSKTLELKVIKENVIQTPAPGPTS (GGGS)1DTSWLKTYSIY GGS EAHKSEIAHRYNDLGEQHFKGLVLIAFSQYLQKCSYDEHAKL italc; MSAVQEVTDFAKTCVADESAANCDKSLHTLFGDKLCAIPNLRENYGELADCCTKQEPER underline;NECFLQHKDDNPSLPPFERPEAEAMCTSFKENPTTFMGHYLHEVARRHPYFYAPEL (GGGS)1LYYAEQYNEILTQCCAEADKESCLTPKLDGVKEKALVSSVRQRMKCSSMQKFGERA italic; IL-2;FKAWAVARLSQTFPNADFAEITKLATDLTKVNKECCHGDLLECADDRAELAKYMCE (H)6 dottedNQATISSKLQTCCDKPLLKKAHCLSEVEHDTMPADLPAIAADFVEDQEVCKNYAEA underline)KDVFLGTFLYEYSRRHPDYSVSLLLRLAKKYEATLEKCCAEANPPACYGTVLAEFQPLVEEPKNLVKTNCDLYEKLGEYGFQNAILVRYTQKAPQVSTPTLVEAARNLGRVGTKCCTLPEDQRLPCVEDYLSAILNRVCLLHEKTPVSEHVTKCCSGSLVERRPCFSALTVDETYVPKEFKAETFTFHSDICTLPEKEKQIKKQTALAELVKHKPKATAEQLKTVMDDFAQFLDTCCKAADKDTCFSTEGPNLVTRCKDALA GGGSAPTSSSTSSSTAEAQQQQQQQQQQQQHLEQLLMDLQELLSRMENYRNLKLPRMLTFKFYLPKQATELKDLQCLEDELGPLRHVLDLTQSKSFQLEDAENFISNIRVTVVKLKGSDNTFECQFDDESATVVDFLRRWIAFCQSIISTSPQ

187 LAIR30.w.A QEGSLPDITIFPNSSLMISQGTFVTVACSYSDKHDLYNMVRLEKGGSTFMEKSTEPYKTEDEFEIGPVNETITGHYSCIYSKGITWSERSKTLELKVIKENVIQTPAPGPTS DTSWLKTYSIY 188LAIR30.w.B QEGSLPDITIFPNSSLMISQGTFVTVVCSYSDKHDLYNMVRLEKDGSTSMEKSTEPYKTEDEFEIGPVNETITGHYSCIYSKGITWSERSKTLELKVTKENVTQTPAPGPTS DTSWLKTYSIY 189LAIR30.w.C QEGSLPDITIFPNSSLMISQGTFVTVVCSYSDKHDLYNMVRLEKDGSTFMGKSTEPYKTEDEFEIGPVNETITGHYSCIYSKGITWSERSKTLELKVIKENVIQTPAPGPTS DTSWLKTYSIY 190LAIR30.w.D QEGSLPDITIFPNSSLMISQGTFVTVVCSYSDKHDLYNMVRLAKDGSTFMEKSTEPYKTEDEFEIGPVNETITGHYSCIYSKGITWSERSKTLELKVIKENVIQTPAPGPTS DTSWLKTYSIY 191LAIR30.w.E QEGSLPDITIFPNSSLMISQGTFVTVVCSYSDKHDLYNMVRLEKDGSTFME

192 LAIR30.w.F QEGSLPDITIFPNSSLMISQGTFVTVVCSYSDKHDLYNMVRLEKDGSTFMEKSTEPYKTEDEFEIGPVNETITGHYSCIYSKGITWSERAKTLELKVIKENVIQTPAPGPTS DTSWLKTYSIY 193LAIR30.2.K1.B QEGSLPDITIFPNSSLVISQGTFVTVVCSYSDKHDLYNMVRLEKDGSTFMEKSTAPYKTEDEFEIGPVNETITGHYSCIYSKGITWSERSKTLELKVIKENVIQTPAPGPTS DTLWLKTYSIY 194LAIR-MSA QEGSLPDITIFPNSSLMISQGTFVTVVCSYSDKHDLYNMVRLEKDGSTFMEKSTEP(LAIR bold; YKTEDEFEIGPVNETITGHYSCIYSKGITWSERSKTLELKVIKENVIQTPAPGPTS(GGGS)1 DTSWLKTYSIY GGSEAHKSEIAHRYNDLGEQHFKGLVLIAFSQYLQKCSYDEHAKLitalic-IL-2- VQEVTDFAKTCVADESAANCDKSLHTLFGDKLCAIPNLRENYGELADCCTKQEPER(H)6 dotted NECFLQHKDDNPSLPPFERPEAEAMCTSFKENPTTFMGHYLHEVARRHPYFYAPELunderline) LYYAEQYNEILTQCCAEADKESCLTPKLDGVKEKALVSSVRQRMKCSSMQKFGERAFKAWAVARLSQTFPNADFAEITKLATDLTKVNKECCHGDLLECADDRAELAKYMCENQATISSKLQTCCDKPLLKKAHCLSEVEHDTMPADLPAIAADFVEDQEVCKNYAEAKDVFLGTFLYEYSRRHPDYSVSLLLRLAKKYEATLEKCCAEANPPACYGTVLAEFQPLVEEPKNLVKTNCDLYEKLGEYGFQNAILVRYTQKAPQVSTPTLVEAARNLGRVGTKCCTLPEDQRLPCVEDYLSAILNRVCLLHEKTPVSEHVTKCCSGSLVERRPCFSALTVDETYVPKEFKAETFTFHSDICTLPEKEKQIKKQTALAELVKHKPKATAEQLKTVMDDFAQFLDTCCKAADKDTCFSTEGPNLVTRCKDALAGGGS

195 4420 HC- DVKLDETGGGLVQPGRPMKLSCVASGFTFSDYWMNWVRQSPEKGLEWVAQIRNKPYLAIR (4420 NYETYYSDSVKGRFTISRDDSKSSVYLQMNNLRVEDMGIYYCTGSYYGMDYWGQGTHC bold; SVTVSATTKGPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVH mIgG1;TFPAVLQSDLYTLSSSVTVPSSTWPSQTVTCNVAHPASSTKVDKKIVPRDCGCKPC (GGGGS)3ICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHT italic; LAIRAQTKPREEQINSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGR underline)PKGGGGSGGGGSGGGGS QEGSLPDITIFPNSSLMISQGTFVTVVCSYSDKHDLYNMVRLEKDGSTFMEKSTEPYKTEDEFEIGPVNETITGHYSCIYSKGITWSERSKTLELKVIKENVIQTPAPGPTSDTSWLKTYSIY 196 3/23 HC-LAIREVQLVESGGGLVQPGRSLKLSCAASGFTLSDYYMAWVRQAPKKGLEWVASINYEGS (3/23 HC bold;STYYGESVKGRFTISRDNAKSTLYLQMNSLRSEDTATYYCVRHDNYFDYWGQGVLV mIgG1;TVSSATTKGPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHT (GGGGS)3FPAVLQSDLYTLSSSVTVPSSTWPSQTVTCNVAHPASSTKVDKKIVPRDCGCKPCI italic; LAIRCTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTA underlineQTKPREEQINSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKGGGGSGGGGSGGGGS QEGSLPDITIFPNSSLMISQGTFVTVVCSYSDKHDLYNMVRLEKDGSTFMEKSTEPYKTEDEFEIGPVNETITGHYSCIYSKGITWSERSKTLELKVIKENVIQTPAPGPTSDTSWLKTYSIY 197 LOB12.3 HC-DVQLVESGGGLVQPGRSLKLSCAASGFIFSYFDMAWVRQAPTKGLEWVASISPDGS LAIRIPYYRDSVKGRFTVSRENAKSSLYLQMDSLRSEDTATYYCARRSYGGYSEIDYWGQ (LOB12.3 HCGVMVTVSSATTKGPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSS bold; mIgG1;GVHTFPAVLQSDLYTLSSSVTVPSSTWPSQTVTCNVAHPASSTKVDKKIVPRDCGC (GGGGS)3KPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVE italic; LAIRVHTAQTKPREEQINSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKT underlineKGRPKGGGGSGGGGSGGGGS QEGSLPDITIFPNSSLMISQGTFVTVVCSYSDKHDLYNMVRLEKDGSTFMEKSTEPYKTEDEFEIGPVNETITGHYSCIYSKGITWSERSKTLELKVIKENVIQTPAPGPTSDTSWLKTYSIY 198QVQLKESGPGLVQPSQTLSLTCTVSGFSLTGYNLHWVRQPPGKGLEWMGRMRYDGDTYYNSVLKSRLSISRDTSKNQVFLKMNSLQTDDTAIYYCTRDGRGDSFDYWGQGVMVTVSSATTKGPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSQTVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTKPREEQINSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKGGGGSGGGGSGGGGS QEGSLPDITIFPNSSLMISQGTFVTVVCSYSDKHDLYNMVRLEKDGSTFMEKSTEPYKTEDEFEIGPV NETITGHYSCIYSKGITWSERSKTLELKVIKENVIQTPAPGPTSDTSWLKTYSIY 199EVQLVESGGGLVQPGKSLKLSCEASGFTFSGYGMHWVRQAPGRGLESVAYITSSSINIKYADAVKGRFTVSRDNAKNLLFLQMNILKSEDTAMYYCARFDWDKNYWGQGTMVTVSSATTKGPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSQTVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTKPREEQINSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKGGGGGGGGGSGGGGG QEGSLPDITIFPNSSLMISQGTFVTVVCSYSDKHDLYNMVRLEKDGSTFMEKSTEPYKTEDEFEIGPVNETITGHYSCIYSKGITWSERSKTLELKVIKENVIQTPAPGPTSDTSWLKTYSIY

indicates data missing or illegible when filed

1. An immunomodulatory fusion protein comprising: (i) animmunomodulatory domain; (ii) a collagen-binding domain, wherein thecollagen-binding domain specifically binds type I and/or type IVcollagen and binds type I collagen with a K_(D)≤500 nM, and wherein thecollagen-binding domain has an isoelectric point pI<10 and a molecularweight (MW) of ≥5 kDa; and (iii) optionally, a linker, wherein theimmunomodulatory domain is operably linked with or without the linker tothe collagen-binding domain. 2-3. (canceled)
 4. The immunomodulatoryfusion protein of claim 1, wherein the collagen-binding domain comprises(i) one or more leucine-rich repeats from a human proteoglycan Class IImember of the small leucine-rich proteoglycan (SLRP) family comprisinglumican, decorin, biglycan, fibromodulin, chondroadherin, asporin,PRELP, osteoadherin/osteomodulin, opticin, osteoglycin/mimecan, podocan,perlecan, and nidogen; or (ii) a human type I glycoprotein having anIg-like domain selected from LAIR1, LAIR2, and Glycoprotein IV, or anextracellular portion thereof which binds collagen, or variants thereof.5-11. (canceled)
 12. The immunomodulatory fusion protein of claim 4,wherein the lumican comprises the amino acid sequence as set forth inSEQ ID NO:
 107. 13-16. (canceled)
 17. The immunomodulatory fusionprotein of claim 4, wherein the human type I glycoprotein is LAIR1 or avariant thereof and the collagen-binding domain comprises (j) amino acidresidues 22-122 of the amino acid sequence as set forth in SEQ ID NO:98, (ii) one or more amino acid substitutions, additions or deletions,optionally two, three, four, five, six, seven, eight, nine, ten or moreamino acid substitutions, additions or deletions relative to a LAIR1protein comprising the amino acid sequence of SEQ ID NO: 98, or (iii) anincreased or decreased binding affinity to collagen relative to acollagen binding affinity of a LAIR1 protein comprising the amino acidsequence of SEQ ID NO:
 98. 18-20. (canceled)
 21. The immunomodulatoryfusion protein of claim 1, wherein the immunomodulatory domain comprisesa polypeptide that (i) activates, enhances or promotes a response by animmune cell, or (ii) inhibits, reduces or suppresses a response by animmune cell.
 22. (canceled)
 23. The immunomodulatory fusion protein ofclaim 21, wherein the immune cell is a lymphoid cell selected from aninnate lymphoid cell, a T cell, a B cell, an NK cell, a monocyte, aneutrophil, a granulocyte, a mast cell, a macrophage, a dendritic cell,or a combination thereof.
 24. (canceled)
 25. The immunomodulatory fusionprotein of claim 21, wherein the response by the immune cell comprisescytokine production, antibody production, production of antigen-specificimmune cells, increased effector function and/or cytotoxicity, or acombination thereof.
 26. The immunomodulatory fusion protein of claim 1,wherein the immunomodulatory domain comprises one or more selected froma cytokine, a chemokine, an activating ligand/receptor, an inhibitoryligand/receptor, an agonist antibody, an antagonist antibody, or acombination thereof.
 27. (canceled)
 28. The immunomodulatory fusionprotein of claim 26, wherein the cytokine is (i) a human gamma commonchain receptor interleukin selected from IL-2, IL-4, IL-7, IL-9, IL-13,IL-15, IL-15/IL-15RA, IL-21, or a combination thereof; (ii) a humanIL-12 family member selected from IL-12 (p35), IL-12 (p40),IL-12(p35)/IL-12(p40), IL-23, IL-27 IL-35, or a combination thereof,(iii) a human IL-1 family member selected from IL-1, IL-18, IL-33, or acombination thereof, or (iv) TNFα, INFα, IFN-γ, GM-CSF, FLT3L, G-CSF,M-CSF, or a combination thereof. 29-34. (canceled)
 35. Theimmunomodulatory fusion protein of claim 26, wherein theimmunomodulatory domain comprises one or more chemokines selected fromLIF, MIP-2, MIP-1α, MIP-1β, CXCL1, CXCL9, CXCL10, MCP-1, Eotaxin,RANTES, LIX, CCL3, CCL4, CCL5, Eotaxin or a combination thereof. 36-37.(canceled)
 38. The immunomodulatory fusion protein of claim 26, whereinthe immunomodulatory domain comprises one or more activatingligands/receptors selected from (i) a TNF superfamily ligand selectedfrom TNF-alpha, CD40L, 4-1BBL, OX40, or an antibody or antigen bindingfragment thereof selected from an anti-TNFR1 antibody, an anti-TNFR2antibody, an anti-CD40 antibody, an anti-4-1BB antibody and an anti-OX40antibody; (ii) a CD28 receptor superfamily or a B7 ligandfamily-selected from ICOS ligand, CD80, CD86, or an antibody or antigenbinding fragment thereof selected from an anti-ICOS antibody and ananti-CD28 antibody; or (iii) a T cell receptor comprising an antibody orantigen binding fragment thereof selected from an anti-CD3γ antibody, ananti-CD3δ antibody, an anti-CD3ζ antibody, and an anti-CD3ε antibody.39-44. (canceled)
 45. The immunomodulatory fusion protein of claim 26,wherein the immunomodulatory domain comprises one or more inhibitoryligands/receptors selected from (i) a CD28 receptor superfamily membercomprising an antibody or antigen binding fragment thereof selected froman anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-PD-L2 antibody,an anti-CTLA4 antibody, (ii) a TNF superfamily member comprises anantibody or antigen binding fragment selected from an anti-TIGITantibody and an anti-BTLA antibody, or (iii) a checkpoint inhibitormember comprising an antibody or antigen binding fragment selected froman anti-VISTA antibody, an anti-TIM-3 antibody, an anti-LAG-3 antibody,an anti-CD47 antibody, and an anti-SIRPα antibody. 46-49. (canceled) 50.The immunomodulatory fusion protein of claim 1, wherein theimmunomodulatory domain is operably linked to the collagen-bindingdomain via a linker of sufficient length or mass to reduce adsorption ofthe immunomodulatory domain onto collagen fibrils, and/or providessufficient molecular weight to the fusion protein reduce diffusion froma tissue, optionally wherein the linker allows for steric separation ofthe immunomodulatory domain from collagen fibrils to promotereceptor/ligand engagement. 51-53. (canceled)
 54. The immunomodulatoryfusion protein of claim 50, wherein the linker (i) is a hydrophilicpolypeptide comprising “N” amino acids in length, wherein 1-1000,10-900, 30-800, 40-700, 50-600, 100-500, or 200-400, (ii) is human serumalbumin or fragment thereof, or (iii) comprises an Fc domain or a mutantFc domain with reduced FcR interaction. 55-56. (canceled)
 57. Theimmunomodulatory fusion protein of claim 1, wherein the fusion proteinis of sufficient mass to reduce size dependent escape by diffusion orconvection upon administration in vivo, optionally wherein the fusionprotein is >60 kDa.
 58. (canceled)
 59. The immunomodulatory fusionprotein of claim 50, wherein the fusion protein binds type I and/or typeIV collagen upon administration in vivo, thereby reducing systemicexposure of the immunomodulatory fusion protein. 60-146. (canceled) 147.A pharmaceutical composition comprising an immunomodulatory fusionprotein of claim 1, and a pharmaceutically acceptable carrier.
 148. Anucleic acid comprising a nucleotide sequence encoding animmunomodulatory fusion protein of claim
 1. 149. An expression vectorcomprising the nucleic acid of claim
 148. 150. A cell transformed withan expression vector of claim
 149. 151. A method for producing animmunomodulatory fusion protein, the method comprising maintaining acell according to claim 150 under conditions permitting expression ofthe immunomodulatory fusion protein.
 152. (canceled)
 153. A method foractivating, enhancing or promoting a response by an immune cell in asubject or inhibiting, reducing or suppressing a response by an immunecell in a subject, comprising administering to a subject in needthereof, an effective amount of the pharmaceutical composition of claim147. 154-158. (canceled)
 159. A method for treating cancer, or reducingor inhibiting tumor growth, comprising administering to a subject inneed thereof, an effective amount of the pharmaceutical composition ofclaim
 147. 160-162. (canceled)
 163. The method of claim 159, whereininfiltration of immune cells into a tumor microenvironment is increasedafter administration of the pharmaceutical composition. 164-166.(canceled)
 167. A kit comprising a container comprising a pharmaceuticalcomposition of claim 147, and a package insert comprising instructionsfor administration of the pharmaceutical composition alone or incombination with another agent, for treating or delaying progression ofcancer or reducing or inhibiting tumor growth in a subject in needthereof. 168-170. (canceled)
 171. A method for reducing or inhibitingtumor growth or treating cancer in a subject, the method comprisingadministering to a subject in need thereof, an effective amount of thepharmaceutical composition of claim 147, and an effective amount of asecond composition comprising (i) a tumor antigen-targeting antibody,optionally wherein the tumor antigen is a tumor-associated antigen(TAA), a tumor-specific antigen (TSA), or a tumor neoantigen and/orwherein the tumor antigen-targeting antibody specifically binds humanHER-2/neu, EGFR, VEGFR, CD20, CD33, CD38 or antigen-binding fragmentthereof, (ii) a cancer vaccine, optionally wherein the cancer vaccine isa peptide comprising one or more tumor-associated antigens, or apopulation of cells immunized in vitro with a tumor antigen andadministered to the subject, (iii) an immune checkpoint inhibitor,optionally comprising an antibody or antigen binding fragment thereofwhich binds PD-1, PD-L1, CTLA-4, LAG3, or TIM3, or (iv) an adoptive celltherapy, optionally comprising an immune effector cell comprising achimeric antigen receptor (CAR) molecule which binds to a tumor antigen,thereby reducing or inhibiting tumor growth or treating cancer in thesubject. 172-176. (canceled)
 177. The method of claim 171, wherein thecancer vaccine is an amphiphilic peptide conjugate comprising atumor-associated antigen, a lipid, and optionally a linker, wherein theamphiphilic peptide conjugate binds albumin under physiologicalconditions, optionally wherein the cancer vaccine further comprises anadjuvant. 178-182. (canceled)
 183. The method of claim 171, wherein theCAR molecule comprises an antigen binding domain, a transmembranedomain, and an intracellular domain comprising a costimulatory domainand/or a primary signaling domain, wherein the antigen binding domainbinds to the tumor antigen associated with the disease, optionallywherein the tumor antigen is selected from CD19, EGFR, Her2/neu, CD30and BCMA. 184-201. (canceled)